Leader H


Full name : Leader Haim N

First name : Haim N

Mail : Weizmann Institute of Science, Department of Materials and Interfaces 76100 Rehovot

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Country : Israel

Email : haimleader@gmail.com

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References (32)

Title : Utilization of diverse organophosphorus pollutants by marine bacteria - Despotovic_2022_Proc.Natl.Acad.Sci.U.S.A_119_e2203604119
Author(s) : Despotovic D , Aharon E , Trofimyuk O , Dubovetskyi A , Cherukuri KP , Ashani Y , Eliason O , Sperfeld M , Leader H , Castelli A , Fumagalli L , Savidor A , Levin Y , Longo LM , Segev E , Tawfik DS
Ref : Proc Natl Acad Sci U S A , 119 :e2203604119 , 2022
Abstract : Anthropogenic organophosphorus compounds (AOPCs), such as phosphotriesters, are used extensively as plasticizers, flame retardants, nerve agents, and pesticides. To date, only a handful of soil bacteria bearing a phosphotriesterase (PTE), the key enzyme in the AOPC degradation pathway, have been identified. Therefore, the extent to which bacteria are capable of utilizing AOPCs as a phosphorus source, and how widespread this adaptation may be, remains unclear. Marine environments with phosphorus limitation and increasing levels of pollution by AOPCs may drive the emergence of PTE activity. Here, we report the utilization of diverse AOPCs by four model marine bacteria and 17 bacterial isolates from the Mediterranean Sea and the Red Sea. To unravel the details of AOPC utilization, two PTEs from marine bacteria were isolated and characterized, with one of the enzymes belonging to a protein family that, to our knowledge, has never before been associated with PTE activity. When expressed in Escherichia coli with a phosphodiesterase, a PTE isolated from a marine bacterium enabled growth on a pesticide analog as the sole phosphorus source. Utilization of AOPCs may provide bacteria a source of phosphorus in depleted environments and offers a prospect for the bioremediation of a pervasive class of anthropogenic pollutants.
ESTHER : Despotovic_2022_Proc.Natl.Acad.Sci.U.S.A_119_e2203604119
PubMedSearch : Despotovic_2022_Proc.Natl.Acad.Sci.U.S.A_119_e2203604119
PubMedID: 35917352

Title : Widespread Utilization of Diverse Organophosphate Pollutants by Marine Bacteria - Despotovic_2021_Biorxiv__
Author(s) : Despotovic D , Aharon E , Trofimyuk O , Dubovetskyi A , Cherukuri KP , Ashani Y , Leader H , Castelli A , Fumagalli A , Savidor A , Levin Y , Longo LM , Segev E , Tawfik DS
Ref : Biorxiv , : , 2020
Abstract : Anthropogenic organophosphates (AOPs), such as phosphotriesters, are used extensively as plasticizers, flame retardants, nerve agents and pesticides. Soil bacteria bearing a phosphotriesterase (PTE) can degrade AOPs, but whether bacteria are capable of utilizing AOPs as a phosphorus source, and how widespread PTEs are in nature, remains unclear. Here, we report the utilization of diverse AOPs by four model marine bacteria and seventeen bacterial isolates from seawater samples. To unravel the details of AOP utilization, two novel PTEs from marine bacteria were isolated and characterized. When expressed in E. coli, these PTEs enabled growth on a pesticide analog as the sole phosphorus source. Utilization of AOPs provides bacteria with a source of phosphorus in depleted environments and offers a new prospect for the bioremediation of a pervasive class of anthropogenic pollutants.
ESTHER : Despotovic_2021_Biorxiv__
PubMedSearch : Despotovic_2021_Biorxiv__

Title : A mixture of three engineered phosphotriesterases enables rapid detoxification of the entire spectrum of known threat nerve agents - Despotovic_2019_Protein.Eng.Des.Sel_32_169
Author(s) : Despotovic D , Aharon E , Dubovetskyi A , Leader H , Ashani Y , Tawfik DS
Ref : Protein Engineering Des Sel , 32 :169 , 2019
Abstract : Nerve agents are organophosphates (OPs) that potently inhibit acetylcholinesterase, and their enzymatic detoxification has been a long-standing goal. Nerve agents vary widely in size, charge, hydrophobicity and the cleavable ester bond. A single enzyme is therefore unlikely to efficiently hydrolyze all agents. Here, we describe a mixture of three previously developed variants of the bacterial phosphotriesterase (Bd-PTE) that are highly stable and nearly sequence identical. This mixture enables effective detoxification of a broad spectrum of known threat agents-GA (tabun), GB (sarin), GD (soman), GF (cyclosarin), VX and Russian-VX. The potential for dimer dissociation and exchange that could inactivate Bd-PTE has minimal impact, and the three enzyme variants are as active in a mixture as they are individually. To our knowledge, this engineered enzyme 'cocktail' comprises the first solution for enzymatic detoxification of the entire range of threat nerve agents.
ESTHER : Despotovic_2019_Protein.Eng.Des.Sel_32_169
PubMedSearch : Despotovic_2019_Protein.Eng.Des.Sel_32_169
PubMedID: 31612205

Title : In vitro evaluation of the catalytic activity of paraoxonases and phosphotriesterases predicts the enzyme circulatory levels required for in vivo protection against organophosphate intoxications - Ashani_2016_Chem.Biol.Interact_259_252
Author(s) : Ashani Y , Leader H , Aggarwal N , Silman I , Worek F , Sussman JL , Goldsmith M
Ref : Chemico-Biological Interactions , 259 :252 , 2016
Abstract : Catalytic scavengers of organophosphates (OPs) are considered very promising antidote candidates for preventing the adverse effects of OP intoxication as stand alone treatments. This study aimed at correlating the in-vivo catalytic efficiency ((kcat/KM)[Enzyme]pl), established prior to the OP challenge, with the severity of symptoms and survival rates of intoxicated animals. The major objective was to apply a theoretical approach to estimate a lower limit for (kcat/KM)[Enzyme]pl that will be adequate for establishing the desired kcat/KM value and plasma concentration of efficacious catalytic bioscavengers. Published data sets by our group and others, from in vivo protection experiments executed in the absence of any supportive medicine, were analyzed. The kcat/KM values of eight OP hydrolyzing enzymes and their plasma concentrations in four species exposed to OPs via s.c., i.m. and oral gavage, were analyzed. Our results show that regardless of the OP type and the animal species employed, sign-free animals were observed following bioscavenger treatment provided the theoretically estimated time period required to detoxify 96% of the OP (t96%) in-vivo was </=10 s. This, for example, can be achieved by an enzyme with kcat/KM = 5 x 107 M-1 min-1 and a plasma concentration of 0.4 muM ((kcat/KM)[Enzyme]pl = 20 min-1). Experiments in which animals were intoxicated by i.v. OP injections did not always conform to this rule, and in some cases resulted in high mortality rates. We suggest that in vivo evaluation of catalytic scavengers should avoid the unrealistic bolus i.v. route of OP exposure.
ESTHER : Ashani_2016_Chem.Biol.Interact_259_252
PubMedSearch : Ashani_2016_Chem.Biol.Interact_259_252
PubMedID: 27163850

Title : Catalytic efficiencies of directly evolved phosphotriesterase variants with structurally different organophosphorus compounds in vitro - Goldsmith_2016_Arch.Toxicol_90_2711
Author(s) : Goldsmith M , Eckstein S , Ashani Y , Greisen P, Jr. , Leader H , Sussman JL , Aggarwal N , Ovchinnikov S , Tawfik DS , Baker D , Thiermann H , Worek F
Ref : Archives of Toxicology , 90 :2711 , 2016
Abstract : The nearly 200,000 fatalities following exposure to organophosphorus (OP) pesticides each year and the omnipresent danger of a terroristic attack with OP nerve agents emphasize the demand for the development of effective OP antidotes. Standard treatments for intoxicated patients with a combination of atropine and an oxime are limited in their efficacy. Thus, research focuses on developing catalytic bioscavengers as an alternative approach using OP-hydrolyzing enzymes such as Brevundimonas diminuta phosphotriesterase (PTE). Recently, a PTE mutant dubbed C23 was engineered, exhibiting reversed stereoselectivity and high catalytic efficiency (k cat/K M) for the hydrolysis of the toxic enantiomers of VX, CVX, and VR. Additionally, C23's ability to prevent systemic toxicity of VX using a low protein dose has been shown in vivo. In this study, the catalytic efficiencies of V-agent hydrolysis by two newly selected PTE variants were determined. Moreover, in order to establish trends in sequence-activity relationships along the pathway of PTE's laboratory evolution, we examined k cat/K M values of several variants with a number of V-type and G-type nerve agents as well as with different OP pesticides. Although none of the new PTE variants exhibited k cat/K M values >107 M-1 min-1 with V-type nerve agents, which is required for effective prophylaxis, they were improved with VR relative to previously evolved variants. The new variants detoxify a broad spectrum of OPs and provide insight into OP hydrolysis and sequence-activity relationships.
ESTHER : Goldsmith_2016_Arch.Toxicol_90_2711
PubMedSearch : Goldsmith_2016_Arch.Toxicol_90_2711
PubMedID: 26612364

Title : Efficacy of the rePON1 mutant IIG1 to prevent cyclosarin toxicity in vivo and to detoxify structurally different nerve agents in vitro - Worek_2014_Arch.Toxicol_88_1257
Author(s) : Worek F , Seeger T , Goldsmith M , Ashani Y , Leader H , Sussman JL , Tawfik DS , Thiermann H , Wille T
Ref : Archives of Toxicology , 88 :1257 , 2014
Abstract : The potent human toxicity of organophosphorus (OP) nerve agents calls for the development of effective antidotes. Standard treatment for nerve agent poisoning with atropine and an oxime has a limited efficacy. An alternative approach is the development of catalytic bioscavengers using OP-hydrolyzing enzymes such as paraoxonases (PON1). Recently, a chimeric PON1 mutant, IIG1, was engineered toward the hydrolysis of the toxic isomers of soman and cyclosarin with high in vitro catalytic efficiency. In order to investigate the suitability of IIG1 as a catalytic bioscavenger, an in vivo guinea pig model was established to determine the protective effect of IIG1 against the highly toxic nerve agent cyclosarin. Prophylactic i.v. injection of IIG1 (1 mg/kg) prevented systemic toxicity in cyclosarin (~2LD50)-poisoned guinea pigs, preserved brain acetylcholinesterase (AChE) activity, and protected erythrocyte AChE activity partially. A lower IIG1 dose (0.2 mg/kg) already prevented mortality and reduced systemic toxicity. IIG1 exhibited a high catalytic efficiency with a homologous series of alkylmethylfluorophosphonates but had low efficiency with the phosphoramidate tabun and was virtually ineffective with the nerve agent VX. This quantitative analysis validated the model for predicting in vivo protection by catalytic bioscavengers based on their catalytic efficiency, the level of circulating enzyme, and the dose of the intoxicating nerve agent. The in vitro and in vivo results indicate that IIG1 may be considered as a promising candidate bioscavenger to protect against the toxic effects of a range of highly toxic nerve agents.
ESTHER : Worek_2014_Arch.Toxicol_88_1257
PubMedSearch : Worek_2014_Arch.Toxicol_88_1257
PubMedID: 24477626

Title : Post-exposure treatment of VX poisoned guinea pigs with the engineered phosphotriesterase mutant C23: A proof-of-concept study - Worek_2014_Toxicol.Lett_231_45
Author(s) : Worek F , Seeger T , Reiter G , Goldsmith M , Ashani Y , Leader H , Sussman JL , Aggarwal N , Thiermann H , Tawfik DS
Ref : Toxicol Lett , 231 :45 , 2014
Abstract : The highly toxic organophosphorus (OP) nerve agent VX is characterized by a remarkable biological persistence which limits the effectiveness of standard treatment with atropine and oximes. Existing OP hydrolyzing enzymes show low activity against VX and hydrolyze preferentially the less toxic P(+)-VX enantiomer. Recently, a phosphotriesterase (PTE) mutant, C23, was engineered towards the hydrolysis of the toxic P(-) isomers of VX and other V-type agents with relatively high in vitro catalytic efficiency (kcat/KM=5x106M-1min-1). To investigate the suitability of the PTE mutant C23 as a catalytic scavenger, an in vivo guinea pig model was established to determine the efficacy of post-exposure treatment with C23 alone against VX intoxication. Injection of C23 (5mgkg-1 i.v.) 5min after s.c. challenge with VX ( approximately 2LD50) prevented systemic toxicity. A lower C23 dose (2mgkg-1) reduced systemic toxicity and prevented mortality. Delayed treatment (i.e., 15min post VX) with 5mgkg-1 C23 resulted in survival of all animals and only in moderate systemic toxicity. Although C23 did not prevent inhibition of erythrocyte acetylcholinesterase (AChE) activity, it partially preserved brain AChE activity. C23 therapy resulted in a rapid decrease of racemic VX blood concentration which was mainly due to the rate of degradation of the toxic P(-)-VX enantiomer that correlates with the C23 blood levels and its kcat/KM value. Although performed under anesthesia, this proof-of-concept study demonstrated for the first time the ability of a catalytic bioscavenger to prevent systemic VX toxicity when given alone as a single post-exposure treatment, and enables an initial assessment of a time window for this approach. In conclusion, the PTE mutant C23 may be considered as a promising starting point for the development of highly effective catalytic bioscavengers for post-exposure treatment of V-agents intoxication.
ESTHER : Worek_2014_Toxicol.Lett_231_45
PubMedSearch : Worek_2014_Toxicol.Lett_231_45
PubMedID: 25195526

Title : Engineering v-type nerve agents detoxifying enzymes using computationally focused libraries - Cherny_2013_ACS.Chem.Biol_8_2394
Author(s) : Cherny I , Greisen P, Jr. , Ashani Y , Khare SD , Oberdorfer G , Leader H , Baker D , Tawfik DS
Ref : ACS Chemical Biology , 8 :2394 , 2013
Abstract : VX and its Russian (RVX) and Chinese (CVX) analogues rapidly inactivate acetylcholinesterase and are the most toxic stockpile nerve agents. These organophosphates have a thiol leaving group with a choline-like moiety and are hydrolyzed very slowly by natural enzymes. We used an integrated computational and experimental approach to increase Brevundimonas diminuta phosphotriesterase's (PTE) detoxification rate of V-agents by 5000-fold. Computational models were built of the complex between PTE and V-agents. On the basis of these models, the active site was redesigned to be complementary in shape to VX and RVX and to include favorable electrostatic interactions with their choline-like leaving group. Small libraries based on designed sequences were constructed. The libraries were screened by a direct assay for V-agent detoxification, as our initial studies showed that colorimetric surrogates fail to report the detoxification rates of the actual agents. The experimental results were fed back to improve the computational models. Overall, five rounds of iterating between experiment and model refinement led to variants that hydrolyze the toxic SP isomers of all three V-agents with kcat/KM values of up to 5 x 10(6) M(-1) min(-1) and also efficiently detoxify G-agents. These new catalysts provide the basis for broad spectrum nerve agent detoxification.
ESTHER : Cherny_2013_ACS.Chem.Biol_8_2394
PubMedSearch : Cherny_2013_ACS.Chem.Biol_8_2394
PubMedID: 24041203

Title : Evolved stereoselective hydrolases for broad-spectrum G-type nerve agent detoxification - Goldsmith_2012_Chem.Biol_19_456
Author(s) : Goldsmith M , Ashani Y , Simo Y , Ben-David M , Leader H , Silman I , Sussman JL , Tawfik DS
Ref : Chemical Biology , 19 :456 , 2012
Abstract : A preferred strategy for preventing nerve agents intoxication is catalytic scavenging by enzymes that hydrolyze them before they reach their targets. Using directed evolution, we simultaneously enhanced the activity of a previously described serum paraoxonase 1 (PON1) variant for hydrolysis of the toxic S(P) isomers of the most threatening G-type nerve agents. The evolved variants show <=340-fold increased rates and catalytic efficiencies of 0.2-5 x 10(7) M(-1) min(-1). Our selection for prevention of acetylcholinesterase inhibition also resulted in the complete reversion of PON1's stereospecificity, from an enantiomeric ratio (E) < 6.3 x 10(-4) in favor of the R(P) isomer of a cyclosarin analog in wild-type PON1, to E > 2,500 for the S(P) isomer in an evolved variant. Given their ability to hydrolyze G-agents, these evolved variants may serve as broad-range G-agent prophylactics.
ESTHER : Goldsmith_2012_Chem.Biol_19_456
PubMedSearch : Goldsmith_2012_Chem.Biol_19_456
PubMedID: 22520752

Title : Computational redesign of a mononuclear zinc metalloenzyme for organophosphate hydrolysis - Khare_2012_Nat.Chem.Biol_8_294
Author(s) : Khare SD , Kipnis Y , Greisen P, Jr. , Takeuchi R , Ashani Y , Goldsmith M , Song Y , Gallaher JL , Silman I , Leader H , Sussman JL , Stoddard BL , Tawfik DS , Baker D
Ref : Nat Chemical Biology , 8 :294 , 2012
Abstract : The ability to redesign enzymes to catalyze noncognate chemical transformations would have wide-ranging applications. We developed a computational method for repurposing the reactivity of metalloenzyme active site functional groups to catalyze new reactions. Using this method, we engineered a zinc-containing mouse adenosine deaminase to catalyze the hydrolysis of a model organophosphate with a catalytic efficiency (k(cat)/K(m)) of ~10(4) M(-1) s(-1) after directed evolution. In the high-resolution crystal structure of the enzyme, all but one of the designed residues adopt the designed conformation. The designed enzyme efficiently catalyzes the hydrolysis of the R(P) isomer of a coumarinyl analog of the nerve agent cyclosarin, and it shows marked substrate selectivity for coumarinyl leaving groups. Computational redesign of native enzyme active sites complements directed evolution methods and offers a general approach for exploring their untapped catalytic potential for new reactivities.
ESTHER : Khare_2012_Nat.Chem.Biol_8_294
PubMedSearch : Khare_2012_Nat.Chem.Biol_8_294
PubMedID: 22306579

Title : Directed evolution of hydrolases for prevention of G-type nerve agent intoxication - Gupta_2011_Nat.Chem.Biol_7_120
Author(s) : Gupta RD , Goldsmith M , Ashani Y , Simo Y , Mullokandov G , Bar H , Ben-David M , Leader H , Margalit R , Silman I , Sussman JL , Tawfik DS
Ref : Nat Chemical Biology , 7 :120 , 2011
Abstract : Organophosphate nerve agents are extremely lethal compounds. Rapid in vivo organophosphate clearance requires bioscavenging enzymes with catalytic efficiencies of >10(7) (M(-1) min(-1)). Although serum paraoxonase (PON1) is a leading candidate for such a treatment, it hydrolyzes the toxic S(p) isomers of G-agents with very slow rates. We improved PON1's catalytic efficiency by combining random and targeted mutagenesis with high-throughput screening using fluorogenic analogs in emulsion compartments. We thereby enhanced PON1's activity toward the coumarin analog of S(p)-cyclosarin by approximately 10(5)-fold. We also developed a direct screen for protection of acetylcholinesterase from inactivation by nerve agents and used it to isolate variants that degrade the toxic isomer of the coumarin analog and cyclosarin itself with k(cat)/K(M) approximately 10(7) M(-1) min(-1). We then demonstrated the in vivo prophylactic activity of an evolved variant. These evolved variants and the newly developed screens provide the basis for engineering PON1 for prophylaxis against other G-type agents.
ESTHER : Gupta_2011_Nat.Chem.Biol_7_120
PubMedSearch : Gupta_2011_Nat.Chem.Biol_7_120
PubMedID: 21217689

Title : In vitro detoxification of cyclosarin in human blood pre-incubated ex vivo with recombinant serum paraoxonases - Ashani_2011_Toxicol.Lett_206_24
Author(s) : Ashani Y , Goldsmith M , Leader H , Silman I , Sussman JL , Tawfik DS
Ref : Toxicol Lett , 206 :24 , 2011
Abstract : An ex vivo protocol was developed to assay the antidotal capacity of rePON1 variants to protect endogenous acetylcholinesterase and butyrylcholinesterase in human whole blood against OP nerve agents. This protocol permitted us to address the relationship between blood rePON1 concentrations, their kinetic parameters, and the level of protection conferred by rePON1 on the cholinesterases in human blood, following a challenge with cyclosarin (GF). The experimental data thus obtained were in good agreement with the predicted percent residual activities of blood cholinesterases calculated on the basis of the rate constants for inhibition of human acetylcholinesterase and butyrylcholinesterase by GF, the concentration of the particular rePON1 variant, and its k(cat)/K(m) value for GF. This protocol thus provides a rapid and reliable ex vivo screening tool for identification of rePON1 bioscavenger candidates suitable for protection of humans against organophosphorus-based toxicants. The results also permitted the refinement of a mathematical model for estimating the efficacious dose of rePON1s variants required for prophylaxis in humans.
ESTHER : Ashani_2011_Toxicol.Lett_206_24
PubMedSearch : Ashani_2011_Toxicol.Lett_206_24
PubMedID: 21807078

Title : Pro-2-PAM therapy for central and peripheral cholinesterases - Demar_2010_Chem.Biol.Interact_187_191
Author(s) : DeMar JC , Clarkson ED , Ratcliffe RH , Campbell AJ , Thangavelu SG , Herdman CA , Leader H , Schulz SM , Marek E , Medynets MA , Ku TC , Evans SA , Khan FA , Owens RR , Nambiar MP , Gordon RK
Ref : Chemico-Biological Interactions , 187 :191 , 2010
Abstract : Novel therapeutics to overcome the toxic effects of organophosphorus (OP) chemical agents are needed due to the documented use of OPs in warfare (e.g. 1980-1988 Iran/Iraq war) and terrorism (e.g. 1995 Tokyo subway attacks). Standard OP exposure therapy in the United States consists of atropine sulfate (to block muscarinic receptors), the acetylcholinesterase (AChE) reactivator (oxime) pralidoxime chloride (2-PAM), and a benzodiazepine anticonvulsant to ameliorate seizures. A major disadvantage is that quaternary nitrogen charged oximes, including 2-PAM, do not cross the blood brain barrier (BBB) to treat brain AChE. Therefore, we have synthesized and evaluated pro-2-PAM (a lipid permeable 2-PAM derivative) that can enter the brain and reactivate CNS AChE, preventing seizures in guinea pigs after exposure to OPs. The protective effects of the pro-2-PAM after OP exposure were shown using (a) surgically implanted radiotelemetry probes for electroencephalogram (EEG), (b) neurohistopathology of brain, (c) cholinesterase activities in the PNS and CNS, and (d) survivability. The PNS oxime 2-PAM was ineffective at reducing seizures/status epilepticus (SE) in diisopropylfluorophosphate (DFP)-exposed animals. In contrast, pro-2-PAM significantly suppressed and then eliminated seizure activity. In OP-exposed guinea pigs, there was a significant reduction in neurological damage with pro-2-PAM but not 2-PAM. Distinct regional areas of the brains showed significantly higher AChE activity 1.5h after OP exposure in pro-2-PAM treated animals compared to the 2-PAM treated ones. However, blood and diaphragm showed similar AChE activities in animals treated with either oxime, as both 2-PAM and pro-2-PAM are PNS active oximes. In conclusion, pro-2-PAM can cross the BBB, is rapidly metabolized inside the brain to 2-PAM, and protects against OP-induced SE through restoration of brain AChE activity. Pro-2-PAM represents the first non-invasive means of administering a CNS therapeutic for the deleterious effects of OP poisoning by reactivating CNS AChE.
ESTHER : Demar_2010_Chem.Biol.Interact_187_191
PubMedSearch : Demar_2010_Chem.Biol.Interact_187_191
PubMedID: 20156430

Title : Stereo-specific synthesis of analogs of nerve agents and their utilization for selection and characterization of paraoxonase (PON1) catalytic scavengers - Ashani_2010_Chem.Biol.Interact_187_362
Author(s) : Ashani Y , Gupta RD , Goldsmith M , Silman I , Sussman JL , Tawfik DS , Leader H
Ref : Chemico-Biological Interactions , 187 :362 , 2010
Abstract : Fluorogenic organophosphate inhibitors of acetylcholinesterase (AChE) homologous in structure to nerve agents provide useful probes for high throughput screening of mammalian paraoxonase (PON1) libraries generated by directed evolution of an engineered PON1 variant with wild-type like specificity (rePON1). Wt PON1 and rePON1 hydrolyze preferentially the less-toxic R(P) enantiomers of nerve agents and of their fluorogenic surrogates containing the fluorescent leaving group, 3-cyano-7-hydroxy-4-methylcoumarin (CHMC). To increase the sensitivity and reliability of the screening protocol so as to directly select rePON1 clones displaying stereo-preference towards the toxic S(P) enantiomer, and to determine accurately K(m) and k(cat) values for the individual isomers, two approaches were used to obtain the corresponding S(P) and R(P) isomers: (a) stereo-specific synthesis of the O-ethyl, O-n-propyl, and O-i-propyl analogs and (b) enzymic resolution of a racemic mixture of O-cyclohexyl methylphosphonylated CHMC. The configurational assignments of the S(P) and R(P) isomers, as well as their optical purity, were established by X-ray diffraction, reaction with sodium fluoride, hydrolysis by selected rePON1 variants, and inhibition of AChE. The S(P) configuration of the tested surrogates was established for the enantiomer with the more potent anti-AChE activity, with S(P)/R(P) inhibition ratios of 10-100, whereas the R(P) isomers of the O-ethyl and O-n-propyl were hydrolyzed by wt rePON1 about 600- and 70-fold faster, respectively, than the S(P) counterpart. Wt rePON1-induced R(P)/S(P) hydrolysis ratios for the O-cyclohexyl and O-i-propyl analogs are estimated to be >>1000. The various S(P) enantiomers of O-alkyl-methylphosphonyl esters of CHMC provide suitable ligands for screening rePON1 libraries, and can expedite identification of variants with enhanced catalytic proficiency towards the toxic nerve agents.
ESTHER : Ashani_2010_Chem.Biol.Interact_187_362
PubMedSearch : Ashani_2010_Chem.Biol.Interact_187_362
PubMedID: 20303930

Title : Two possible orientations of the HI-6 molecule in the reactivation of organophosphate-inhibited acetylcholinesterase. -
Author(s) : Luo C , Leader H , Radic Z , Maxwell DM , Taylor P , Doctor BP , Saxena A
Ref : Cholinergic Mechanisms, CRC Press :627 , 2004

Title : Two possible orientations of the HI-6 molecule in the reactivation of organophosphate-inhibited acetylcholinesterase - Luo_2003_Biochem.Pharmacol_66_387
Author(s) : Luo C , Leader H , Radic Z , Maxwell DM , Taylor P , Doctor BP , Saxena A
Ref : Biochemical Pharmacology , 66 :387 , 2003
Abstract : The inhibition of acetylcholinesterase (AChE) by organophosphorus compounds (OPs) causes acute toxicity or death of the intoxicated individual. One group of these compounds, the OP nerve agents, pose an increasing threat in the world due to their possible use in the battlefield or terrorist acts. Antidotes containing oxime compounds to reactivate the inhibited enzyme are highly valued for treatment against OP poisoning. One of these reactivators, HI-6, was shown to be significantly more effective in treating soman toxicity than other oximes, such as 2-PAM, TMB4, and obidoxime. However, HI-6 was less effective in reactivating AChE inhibited by the OP pesticide, paraoxon. In this study, the mechanism for HI-6-induced reactivation of OP-AChE conjugates was investigated using mouse mutant AChEs inhibited with different OPs including organophosphate paraoxon, and several methylphosphonates. Results indicate that the HI-6 molecule may assume two different orientations in the reactivation of AChE inhibited by organophosphate and Sp methylphosphonates. These conclusions were further corroborated by reactivation studies using an analog of HI-6 in which the bispyridinium moieties are linked by a methylene bridge rather than an ether oxygen.
ESTHER : Luo_2003_Biochem.Pharmacol_66_387
PubMedSearch : Luo_2003_Biochem.Pharmacol_66_387
PubMedID: 12907237

Title : Inhibition of cholinesterases with cationic phosphonyl oximes highlights distinctive properties of the charged pyridine groups of quaternary oxime reactivators - Ashani_2003_Biochem.Pharmacol_66_191
Author(s) : Ashani Y , Bhattacharjee AK , Leader H , Saxena A , Doctor BP
Ref : Biochemical Pharmacology , 66 :191 , 2003
Abstract : Oxime-induced reactivation of phosphonylated cholinesterases (ChEs) produces charged phosphonyl pyridine oxime intermediates (POXs) that are most potent organophosphate (OP) inhibitors of ChEs. To understand the role of cationic pyridine oxime leaving groups in the enhanced anti-ChE activity of POXs, the bimolecular rate constants for the inhibition (k(i)) of acetylcholinesterases (AChE) and butyrylcholinesterases (BChE), and the rate of decomposition (k(d)) of authentic O-alkyl methylphosphonyl pyridine oximes (AlkMeP-POXs) and N,N-dimethylamidophosphoryl pyridine oximes (EDMP-POXs), were studied. Stability ranking order in aqueous solutions correlated well with the electronic features and optimized geometries that were obtained by ab initio calculations at 6-31G(**) basis set level. AlkMeP-POXs of the 2-pyridine oxime series were found to be 4- to 8-fold more stable (t(1/2)=0.7 to 1.5 min) than the homologous O,O-diethylphosphoryl (DEP) oxime. Results suggest that re-inhibition of enzyme activity by POX is less likely during the reactivation of DEP-ChEs (obtained by use of DEP-containing pesticides) by certain oximes, compared to nerve agent-inhibited ChEs. The greatest inhibition was observed for the O-cyclohexyl methylphosphonyl-2PAM derivative (4.0 x 10(9)M(-1)min(-1); mouse AChE) and is 10-fold higher than the k(i) of cyclosarin. Increasing the size of the O-alkyl substituent of AlkMeP-POXs had only a small to moderate effect on the k(i) of ChEs, signifying a major role for the cationic pyridine oxime leaving group in the inhibition reaction. The shape of plots of logk(i) vs. pK(a) of the leaving groups for AlkMeP-PAMs and DEP-PAMs, could be used as a diagnostic tool to highlight and rationalize the unique properties of the cationic moiety of pyridine oxime reactivators.
ESTHER : Ashani_2003_Biochem.Pharmacol_66_191
PubMedSearch : Ashani_2003_Biochem.Pharmacol_66_191
PubMedID: 12826262

Title : Pyridophens: binary pyridostigmine-aprophen prodrugs with differential inhibition of acetylcholinesterase, butyrylcholinesterase, and muscarinic receptors - Leader_2002_J.Med.Chem_45_902
Author(s) : Leader H , Wolfe AD , Chiang PK , Gordon RK
Ref : Journal of Medicinal Chemistry , 45 :902 , 2002
Abstract : A series of "binary prodrugs" called carbaphens,(1) carbamylated derivatives on one or both of the aromatic rings of the muscarinic receptor antagonist aprophen [(N,N-diethylamino)ethyl 2,2-diphenylpropionate], were synthesized to develop binary prophylactic agents against organophosphorus intoxication. As a group, the carbaphens retained the muscarinic receptor antagonist properties of aprophen but also preferentially inhibited butyrylcholinesterase (BChE) in contrast to acetylcholinesterase (AChE). Therefore, a new series of compounds named pyridophens were designed and synthesized to achieve binary prodrugs to preferentially inhibit AChE over BChE, while still retaining the muscarinic receptor antagonism of aprophen. The pyridophens consist of the basic pyridostigmine skeleton combined with the 2,2-diphenylpropionate portion of aprophen by replacement of the diethylamino group. Three compounds, 9 (a tertiary pyridine), 10 (a quaternary pyridine), and 12 (a tertiary tetrahydropyridine), were found to be effective inhibitors of both BChE and AChE. However, 10, N-methyl-3-[[(dimethylamino)carbonyl]oxy]-2-(2'2'-diphenylpropionoxy-methyl)pyridinium iodide, inhibited AChE selectively over BChE, with a bimolecular rate constant similar to pyridostigmine. In contrast to their potent cholinesterase inhibitory activity, all of the pyridophen analogues were less potent antagonists of the muscarinic receptor than aprophen.
ESTHER : Leader_2002_J.Med.Chem_45_902
PubMedSearch : Leader_2002_J.Med.Chem_45_902
PubMedID: 11831902

Title : Role of edrophonium in prevention of the re-inhibition of acetylcholinesterase by phosphorylated oxime - Luo_1999_Chem.Biol.Interact_119-120_129
Author(s) : Luo C , Saxena A , Ashani Y , Leader H , Radic Z , Taylor P , Doctor BP
Ref : Chemico-Biological Interactions , 119-120 :129 , 1999
Abstract : We examined the role of edrophonium in the acceleration phenomenon using mouse wild-type and mutant D74N AChE inhibited with 7-(O,O-diethyl-phosphinyloxy)-1-methylquinolinium methylsulfate (DEPQ). With DEPQ-inhibited wild-type mouse acetylcholinesterase (AChE), the reactivation kinetic profile demonstrated one-phase exponential association only when 2-[hydroxyimino methyl]-1-methylpyridinium chloride (2-PAM) and 1-(2-hydroxy-iminomethyl-1-pyridinium)-1-(4-carboxy-aminopyridi nium)-dimethyl ether hydrochloride (HI-6) were used as reactivators. When 1,1[oxybis-methylene)bis[4-(hydroxyimino)methyl] pyridinium dichloride (LuH6) and 1,1-trimethylene bis(4-hydroxyimino methyl) pyridinium dichloride (TMB4) were used, the reactivation kinetic profile was biphasic in nature. Edrophonium had no effect on reactivation by 2-PAM and HI-6, but significantly accelerated LuH6- and TMB4-induced reactivation of DEPQ-inhibited wild-type mouse AChE. Comparison of the initial and overall reactivation rate constants with five oximes indicated that acceleration by edrophonium may be due to the prevention of re-inhibition of the reactivated enzyme by the phosphorylated oxime (POX) produced during the reactivation. With LuH6 and TMB4, about 2.5-fold increase in the reactivation rate constants was observed in the presence of edrophonium, but little or no effect was observed with the other three oximes. The initial reactivation rate constants were 5.4- and 4.2-fold of the overall rate constants with LuH6 and TMB4 as reactivators respectively, however, very little change was found between the initial and overall rate constants with the other three oximes. In experiments with D74N AChE, for which the inhibition potency of charged organophosphate (OP) was two to three orders less than wild-type enzyme, edrophonium had no effect on the reactivation by LuH6 and TMB4 and the time courses of reactivation were monophasic. The data from mutant enzyme substantiate the involvement of edrophonium in protecting POX re-inhibition of reactivated enzyme formed during the reactivation of OP-inhibited AChE.
ESTHER : Luo_1999_Chem.Biol.Interact_119-120_129
PubMedSearch : Luo_1999_Chem.Biol.Interact_119-120_129
PubMedID: 10421446

Title : Characterization of O,O-diethylphosphoryl oximes as inhibitors of cholinesterases and substrates of phosphotriesterases - Leader_1999_Biochem.Pharmacol_58_503
Author(s) : Leader H , Vincze A , Manisterski B , Rothschild N , Dosoretz C , Ashani Y
Ref : Biochemical Pharmacology , 58 :503 , 1999
Abstract : Reactivators of organophosphate (OP)-inhibited cholinesterases (ChEs) are believed to give rise to phosphorylated oximes (POX) that reinhibit the enzyme. Diethylphosphoryl oximes (DEP-OX) that were generated in situ were demonstrated in the past to be unstable, yet were more potent inhibitors of acetylcholinesterase (AChE) than the parent OPs. In view of the inconsistencies among reported results, and the potential toxicity of POXs, it seemed important to characterize authentic DEP-OXs, and to evaluate their interference with reactivation of diethylphosphoryl-ChE (DEP-ChE) conjugates. To this end, the diethylphosphoric acid esters of 1-methyl-2-pyridinium carboxaldehyde oxime (DEP-2PAM) and 1-methyl-4 pyridinium carboxaldehyde oxime (DEP-4PAM) were synthesized and chemically defined. The half-lives of DEP-2PAM and DEP-4PAM in 10 mM Tris buffer, pH 7.8, at 29 degrees were found to be 10 and 980 sec, respectively. The two DEP-OXs inhibited ChEs with the following ranking order: for DEP-2PAM, human butyrylcholinesterase (HuBChE, k(i) = 2.03 x 10(9) M(-1) min(-1)) > mouse AChE (MoAChE) approximately equal to fetal bovine serum AChE (FBS-AChE) approximately equal to equine BChE (EqBChE); for DEP-4PAM, HuBChE (k(i) = 0.71 x 10(9) M(-1) min(-1)) > EqBChE > MoAChE > FBS-AChE. A dialkylarylphosphate hydrolase (phosphotriesterase; PTE) from Pseudomonas sp. catalyzed the hydrolysis of DEP-4PAM with k(cat)/Km = 3.56 x 10(7) M(-1) min(-1) and Km = 0.78 mM. Reactivation of DEP-ChEs was enhanced by PTE when 4-PAM-based oximes were used as reactivators, whereas reactivation with 2-PAM-based oximes was not affected by PTE. This observation is attributed primarily to the short half-life of DEP-OXs derived from the latter oximes. Relatively low doses of PTE can detoxify large quantities of DEP-OXs rapidly, and thereby augment the efficacy of antidotes that contain the oxime function in position 4 of the pyridine ring.
ESTHER : Leader_1999_Biochem.Pharmacol_58_503
PubMedSearch : Leader_1999_Biochem.Pharmacol_58_503
PubMedID: 10424771

Title : Sequestration of Toxic Phosphorylated Oximes by Stoichiometric and Catalytic Scavengers -
Author(s) : Leader H , Vincze A , Rothschild N , Dosoretz C , Ashani Y
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :305 , 1998

Title : Oxidative biodegradation of phosphorothiolates by fungal laccase - Amitai_1998_FEBS.Lett_438_195
Author(s) : Amitai G , Adani R , Sod-Moriah G , Rabinovitz I , Vincze A , Leader H , Chefetz B , Leibovitz-Persky L , Friesem D , Hadar Y
Ref : FEBS Letters , 438 :195 , 1998
Abstract : Organophosphorus (OP) insecticides and nerve agents that contain P-S bond are relatively more resistant to enzymatic hydrolysis. Purified phenol oxidase (laccase) from the white rot fungus Pleurotus ostreatus (Po) together with the mediator 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) displayed complete and rapid oxidative degradation of the nerve agents VX and Russian VX (RVX) and the insecticide analog diisopropyl-Amiton with specific activity: k(sp) = 2200, 667 and 1833 nmol min(-1) mg(-1), respectively (pH 7.4, 37 degrees C). A molar ratio of 1:20 for OP/ABTS and 0.05 M phosphate at pH 7.4 provided the highest degradation rate of VX and RVX. The thermostable laccase purified from the fungus Chaetomium thermophilium (Ct) in the presence of ABTS caused a 52-fold slower degradation of VX with k(sp) = 42 nmol min(-1) mg(-1). The enzymatic biodegradation products were identified by 31P-NMR and GC/MS analysis.
ESTHER : Amitai_1998_FEBS.Lett_438_195
PubMedSearch : Amitai_1998_FEBS.Lett_438_195
PubMedID: 9827544

Title : Acceleration of Oxime-Induced Reactivation of Organophosphate-Inhibited Acetylcholinesterase by Quaternary Ligands -
Author(s) : Luo C , Ashani Y , Saxena A , Leader H , Maxwell DM , Taylor P , Doctor BP
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :215 , 1998

Title : Combined effect of organophosphorus hydrolase and oxime on the reactivation rate of diethylphosphoryl-acetylcholinesterase conjugates - Ashani_1998_Biochem.Pharmacol_55_159
Author(s) : Ashani Y , Leader H , Rothschild N , Dosoretz C
Ref : Biochemical Pharmacology , 55 :159 , 1998
Abstract : Reactivation of inhibited acetylcholinesterase (AChE) is essential for rapid recovery after organophosphate (OP) poisoning. However, following administration of an oxime reactivator, such as pralidoxime mesylate (P2S), in patients poisoned with certain diethylphosphorothioate pesticides, no reactivation is observed, presumably due to reinhibition by circulating anti-cholinesterase OPs. Pretreatment alone with organophosphorus hydrolases (OPH) that are capable of rapidly hydrolyzing OPs was demonstrated, in animals, to confer significant protection against OP toxicity. One strategy to augment the potentially therapeutic scope of OPHs is a combined post-exposure treatment consisting of a drug(s) commonly used against OP toxicity and a suitable hydrolase. In this study, we examined the in vitro ability of OPH from Pseudomonas sp. (OPHps) to prevent reinhibition of P2S-reactivated AChE by excess OPs. The kinetic parameters of the reactivation of a series of diethylphosphoryl-AChE (DEP--AChE) conjugates, obtained by the use of various diethylphosphates, were determined and compared with the rates of reactivation in the presence of OPHps, with and without the OP inhibitors in the reactivation medium. Extrapolation of the in vitro results to in vivo conditions suggests that an OPHps concentration as low as 1 microgram/mL blood would result in a 100-fold decrease in the concentration of circulating anti-AChE pesticides within less than one blood-circulation time, thereby minimizing reinhibition of the reactivated enzyme. Thus, for DEP-based pesticides, the combination of P2S-OPH treatment can significantly improve clinical recovery after OP intoxication. In addition, it is shown here for the first time that an OPH can effectively hydrolyze quaternary ammonium-containing OPs. This indicates that hydrolysis of phosphorylated oximes, toxic side products of oxime treatment, may also be accelerated by OPHs.
ESTHER : Ashani_1998_Biochem.Pharmacol_55_159
PubMedSearch : Ashani_1998_Biochem.Pharmacol_55_159
PubMedID: 9448738

Title : Synthesis and antimuscarinic activity of 2-[N-(ethyl)-(N-beta- hydroxyethyl)]aminoethyl 2,2-diphenylpropionate, a metabolite of aprophen - Brown_1993_J.Pharm.Sci_82_563
Author(s) : Brown ND , Leader H , Phillips LR , Smejkal RM , Gordon RK , Chiang PK
Ref : J Pharm Sci , 82 :563 , 1993
Abstract : The preparation of 2-[N-(ethyl)-(N-beta-hydroxyethyl)]amino-ethyl 2,2-diphenylpropionate (1), a metabolite of aprophen [2-diethylaminoethyl 2,2-diphenylpropionate], is described. Hydrolysis of [2-(2-chloroethyl)ethylamino]ethyl acetate hydrochloride (2) in a basic solution, followed by acidic pH adjustment, gave the ethylcholineaziridinium ion (3) that upon treatment with 2,2-diphenylpropionic acid produced 1 in a 56% yield. Synthetic 1 was found to possess antimuscarinic activities, but was approximately 10-fold less potent than the parent compound aprophen.
ESTHER : Brown_1993_J.Pharm.Sci_82_563
PubMedSearch : Brown_1993_J.Pharm.Sci_82_563
PubMedID: 8331525

Title : Muscarinic receptor subtype specificity of (N,N-dialkylamino)alkyl 2- cyclohexyl-2-phenylpropionates: cylexphenes (cyclohexyl-substituted aprophen analogues) - Leader_1992_J.Med.Chem_35_1290
Author(s) : Leader H , Gordon RK , Baumgold J , Boyd VL , Newman AH , Smejkal RM , Chiang PK
Ref : Journal of Medicinal Chemistry , 35 :1290 , 1992
Abstract : A series of aprophen [(N,N-diethylamino)ethyl 2,2-diphenylpropionate] analogues, called cylexphenes, were synthesized with alterations in (1) the chain length of the amine portion of the ester, (2) the alkyl groups on the amino alcohol, and (3) a cyclohexyl group replacing one of the phenyl rings. The antimuscarinic activities of these analogues were assessed in two pharmacological assays: the inhibition of acetylcholine-induced contraction of guinea pig ileum, and the blocking of carbachol-stimulated release of alpha-amylase from rat pancreatic acinar cells. These two tissues represent the M3(ileum) and M3(pancreas) muscarinic receptor subtypes. In addition, the analogues were also evaluated for their competitive inhibition of the binding of [3H]NMS to selected cell membranes, each containing only one of the m1, M2, m3, or M4 muscarinic receptor subtypes. The m1 and m3 receptors were stably transfected into A9 L cells. The replacement of one phenyl group of aprophen with a cyclohexyl group increased the selectivity of all the analogues for the pancreatic acinar muscarinic receptor subtype over the ileum subtype by more than 10-fold, with the (N,N-dimethylamino)propyl analogue exhibiting the greatest selectivity for the pancreas receptor subtype, over 30-fold. The cylexphenes also showed a decrease in potency in comparison to the parent compound when examined for the binding of [3H]NMS to the M2 subtype. In agreement with the pharmacological data obtained from the pancreas, the (N,N-dimethylamino)propyl cylexphene 3 demonstrated the greatest selectivity for the m3 subtype, and additionally showed a preference for the m1 and M4 receptor subtypes over the M2 receptor subtype in the binding assay. Thus, this compound showed a potent selectivity according to the pharmacological and binding assays between the muscarinic receptor subtypes of the pancreas and ileum. In both the pharmacological and binding assays, the potency of the analogues decreased markedly when the chain length and the bond distance between the carbonyl oxygen and protonated nitrogen were increased beyond three methylene groups. When the structures of these analogues were analyzed using a molecular modeling program, the bond distance between the carbonyl oxygen and protonated nitrogen was deduced to be more important for the antagonist activity than subtype specificity.
ESTHER : Leader_1992_J.Med.Chem_35_1290
PubMedSearch : Leader_1992_J.Med.Chem_35_1290
PubMedID: 1560441

Title : Protection against tabun toxicity in mice by prophylaxis with an enzyme hydrolyzing organophosphate esters - Raveh_1992_Biochem.Pharmacol_44_397
Author(s) : Raveh L , Segall Y , Leader H , Rothschild N , Levanon D , Henis Y , Ashani Y
Ref : Biochemical Pharmacology , 44 :397 , 1992
Abstract : We demonstrate here the correlation between protection afforded by pretreatment alone with parathion hydrolase purified from Pseudomonas sp. against tabun toxicity in mice and the kinetic parameters which are assumed to determine the in vivo detoxification of tabun by the same enzyme. Results show that 15 and 22 micrograms of parathion hydrolase per animal conferred a protective ratio of 3.94 and 5.65 respectively, against tabun toxicity, without post-exposure treatment.
ESTHER : Raveh_1992_Biochem.Pharmacol_44_397
PubMedSearch : Raveh_1992_Biochem.Pharmacol_44_397
PubMedID: 1322669

Title : Isolation and identification of beta-hydroxyethylaprophen: a urinary metabolite of aprophen in rats - Brown_1991_J.Chromatogr_563_466
Author(s) : Brown ND , Phillips LR , Leader H , Chiang PK
Ref : Journal of Chromatography , 563 :466 , 1991
Abstract : The metabolism of the anticholinergic drug aprophen was studied in rats after oral administration via stomach intubation. beta-Hydroxyethylaprophen, a major urinary metabolite of aprophen, was isolated and identified by normal-phase high-performance liquid chromatography and electron ionization mass spectrometry. More than 22% of the parent drug was recovered and quantified over a 72-h collection period. Results show that 2,2-diphenylpropionic acid, another major metabolite of aprophen which lacks anticholinergic properties, was also isolated and identified in this study. Experiments are currently underway to synthesize and test the anticholinergic properties of beta-hydroxyethylaprophen in mammals.
ESTHER : Brown_1991_J.Chromatogr_563_466
PubMedSearch : Brown_1991_J.Chromatogr_563_466
PubMedID: 2056011

Title : Aprophit: an irreversible antagonist for muscarinic receptors - Newman_1990_Biochem.Pharmacol_40_1357
Author(s) : Newman AH , Covington J , Oleshansky M , Jackson BW , Weissman BA , Leader H , Chiang PK
Ref : Biochemical Pharmacology , 40 :1357 , 1990
Abstract : The development of selective irreversible ligands has proven to be an invaluable technique for the isolation, purification and characterization of many receptor proteins. An isothiocyanato-derivative of the muscarinic antagonist aprophen was synthesized and evaluated as a potential irreversible ligand for muscarinic receptors. This compound (aprophit) displaced [3H]N-methylscopolamine binding from rat cerebral cortex with a Ki of 3.1 x 10(-7) M. The inhibition was concentration-dependent and could not be reversed by extensive washing. Aprophit inhibited the acetylcholine-stimulated release of catecholamines from isolated, perfused guinea pig adrenal glands in a concentration-dependent manner. This inhibition was not reversed by perfusing the tissue with Locke's solution and was not due to a non-selective acylation by the isothiocyanate function. The data suggest that aprophit is selectively acylating muscarinic receptor proteins and thus may be useful in their further characterization.
ESTHER : Newman_1990_Biochem.Pharmacol_40_1357
PubMedSearch : Newman_1990_Biochem.Pharmacol_40_1357
PubMedID: 2403389

Title : Binary antidotes for organophosphate poisoning: aprophen analogues that are both antimuscarinics and carbamates - Leader_1989_J.Med.Chem_32_1522
Author(s) : Leader H , Smejkal RM , Payne CS , Padilla FN , Doctor BP , Gordon RK , Chiang PK
Ref : Journal of Medicinal Chemistry , 32 :1522 , 1989
Abstract : Prophylaxis against organophosphate poisoning can be achieved by pretreatment with physostigmine or pyridostigmine, which are carbamates, and aprophen, which is an anticholinergic agent. Thus, a series of aprophen analogues was synthesized with carbamyl substitutions on the phenyl rings (carbaphens). The rationale behind this design is that such compounds might exhibit most of the therapeutic characteristics of aprophen, as well as the ability to protect prophylactically by chemically masking cholinesterase enzymes. Compounds 4 (dimethylhydroxycarbaphen), 15 (dimethylcarbaphen), and 16 (monomethylcarbaphen) were found to inactivate human butyrylcholinesterase in a time-dependent manner with potencies similar to those of physostigmine or pyridostigmine, and the latter two exhibited almost the same antimuscarinic profile as aprophen. In contrast to the potent inactivation of butyrylcholinesterase by these compounds, marginal inactivation of acetylcholinesterase activity was observed, and only at much higher drug concentrations. The noncarbamylated analogues had no effect on the activity of either cholinesterase. The carbaphen compounds are hence prototype drugs that can interact with either muscarinic receptors or butyrylcholinesterase. Furthermore, these compounds are prodrugs, since after carbamylation of the cholinesterase, the leaving group 14 (hydroxyaprophen) is a potent antimuscarinic itself.
ESTHER : Leader_1989_J.Med.Chem_32_1522
PubMedSearch : Leader_1989_J.Med.Chem_32_1522
PubMedID: 2738887

Title : In vitro and in vivo protection of acetylcholinesterase against organophosphate poisoning by pretreatment with a novel derivative of 1,3,2-dioxaphosphorinane 2-oxide - Ashani_1983_J.Med.Chem_26_145
Author(s) : Ashani Y , Leader H , Raveh L , Bruckstein R , Spiegelstein M
Ref : Journal of Medicinal Chemistry , 26 :145 , 1983
Abstract : Covalent molecular combinations of a cyclic phosphate (dioxaphosphorinane) and a potential leaving group, such as 3-(trimethylammonio)phenol iodide (TMPH), suggested the synthesis of O-[3-(trimethylammonio)phenyl]-1,3,2-dioxaphosphorinane 2-oxide iodide (TDPI). TDPI inhibited acetylcholinesterase (AChE) (ki = 8.4 x 10(3) M-1 min-1) via the formation of an unstable covalent intermediate. TDPI-inhibited AChE hydrolyzed spontaneously with t1/2 approximately equal to 10 min. Butyrylcholinesterase (BuChE) was also inhibited by TDPI (ki = 1.8 x 10(4) M-1 min-1), but the inhibited BuChE was more stable (greater than 10 times) than the corresponding AchE-TDPI conjugate. Pretreatment of mice with TDPI conferred protection against 22 LD50's of paraoxon and 5 LD50's of soman, provided that treatment with anticholinergics and an oxime followed administration of these anticholinesterase poisons. Correlation between in vitro and in vivo observations suggests that the main protection of AChE conferred by TDPI results from temporary masking of the active site of the enzyme. The acute toxicity of TDPI was found to be 444 mg/kg (sc, mice), whereas analogous carbamates and a noncyclic phosphate also displaying antidotal properties are greater than 170 times more toxic.
ESTHER : Ashani_1983_J.Med.Chem_26_145
PubMedSearch : Ashani_1983_J.Med.Chem_26_145
PubMedID: 6827532

Title : Sugar-oximes, new potential antidotes against organophosphorus poisoning - Rachaman_1979_Arzneimittelforschung_29_875
Author(s) : Rachaman ES , Ashani Y , Leader H , Granoth I , Edery H , Porath G
Ref : Arzneimittelforschung , 29 :875 , 1979
Abstract : In attempt to improve distribution and transport qualities of antidotes against organophosphorus poisoning, a new series of pyridine aldoximes linked to glucose moiety were synthesized and studied both in vivo and in vitro. Preliminary results describing the biological activity of the new compounds are presented and discussed in this report.
ESTHER : Rachaman_1979_Arzneimittelforschung_29_875
PubMedSearch : Rachaman_1979_Arzneimittelforschung_29_875
PubMedID: 582776