Goldsmith M

General

Full name : Goldsmith Moshe

First name : Moshe

Mail : Weizmann Institute of Science\; Biological Chemistry\; Herzel st.\; Rehovot\; 76100

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Country : Israel

Email : moshe.goldsmith@weizmann.ac.il

Phone : +972523324675

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References (16)

Title : Catalytic bioscavengers as countermeasures against organophosphate nerve agents - Goldsmith_2018_Chem.Biol.Interact_292_50
Author(s) : Goldsmith M , Ashani Y
Ref : Chemico-Biological Interactions , 292 :50 , 2018
Abstract : Recent years have seen an increasing number of incidence, in which organophosphate nerve agents (OPNAs) have been used against civilians with devastating outcomes. Current medical countermeasures against OPNA intoxications are aimed at mitigating their symptoms, but are unable to effectively prevent them. In addition, they may fail to prevent the onset of a cholinergic crisis in the brain and its secondary toxic manifestations. The need for improved medical countermeasures has led to the development of bioscavengers; proteins and enzymes that may prevent intoxication by binding and inactivating OPNAs before they can reach their target organs. Non-catalytic bioscavengers such as butyrylcholinesterase, can rapidly bind OPNA molecules in a stoichiometric and irreversible manner, but require the administration of large protein doses to prevent intoxication. Thus, many efforts have been made to develop catalytic bioscavengers that could rapidly detoxify OPNAs without being inactivated in the process. Such enzymes may provide effective prophylactic protection and improve post-exposure treatments using much lower protein doses. Here we review attempts to develop catalytic bioscavengers using molecular biology, directed evolution and enzyme engineering techniques; and natural or computationally designed enzymes. These include both stoichiometric scavengers and enzymes that can hydrolyze OPNAs with low catalytic efficiencies. We discuss the catalytic parameters of evolved and engineered enzymes and the results of in-vivo protection and post-exposure experiments performed using OPNAs and bioscavengers. Finally, we briefly address some of the challenges that need to be met in order to transition these enzymes into clinically approved drugs.
ESTHER : Goldsmith_2018_Chem.Biol.Interact_292_50
PubMedSearch : Goldsmith_2018_Chem.Biol.Interact_292_50
PubMedID: 29990481

Title : Single treatment of VX poisoned guinea pigs with the phosphotriesterase mutant C23AL: Intraosseous versus intravenous injection - Wille_2016_Toxicol.Lett_258_198
Author(s) : Wille T , Neumaier K , Koller M , Ehinger C , Aggarwal N , Ashani Y , Goldsmith M , Sussman JL , Tawfik DS , Thiermann H , Worek F
Ref : Toxicol Lett , 258 :198 , 2016
Abstract : The recent attacks with the nerve agent sarin in Syria reveal the necessity of effective countermeasures against highly toxic organophosphorus compounds. Multiple studies provide evidence that a rapid onset of antidotal therapy might be life-saving but current standard antidotal protocols comprising reactivators and competitive muscarinic antagonists show a limited efficacy for several nerve agents. We here set out to test the newly developed phosphotriesterase (PTE) mutant C23AL by intravenous (i.v.), intramuscular (i.m.; model for autoinjector) and intraosseous (i.o.; model for intraosseous insertion device) application in an in vivo guinea pig model after VX challenge ( approximately 2LD50). C23AL showed a Cmax of 0.63mumolL(-1) after i.o. and i.v. administration of 2mgkg(-1) providing a stable plasma profile up to 180min experimental duration with 0.41 and 0.37mumolL(-1) respectively. The i.m. application of C23AL did not result in detectable plasma levels. All animals challenged with VX and subsequent i.o. or i.v. C23AL therapy survived although an in part substantial inhibition of erythrocyte, brain and diaphragm AChE was detected. Theoretical calculation of the time required to hydrolyze in vivo 96.75% of the toxic VX enantiomer is consistent with previous studies wherein similar activity of plasma containing catalytic scavengers of OPs resulted in non-lethal protection although accompanied with a variable severity of cholinergic symptoms. The relatively low C23AL plasma level observed immediately after its i.v. or i.o load, point at a possible volume of distribution greater than the guinea pig plasma content, and thus underlines the necessity of in vivo experiments in antidote research. In conclusion the i.o. application of PTE is efficient and resulted in comparable plasma levels to the i.v. application at a given time. Thus, i.o. vascular access systems could improve the post-exposure PTE therapy of nerve agent poisoning.
ESTHER : Wille_2016_Toxicol.Lett_258_198
PubMedSearch : Wille_2016_Toxicol.Lett_258_198
PubMedID: 27397758

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 : Automated Structure- and Sequence-Based Design of Proteins for High Bacterial Expression and Stability - Goldenzweig_2016_Mol.Cell_63_337
Author(s) : Goldenzweig A , Goldsmith M , Hill SE , Gertman O , Laurino P , Ashani Y , Dym O , Unger T , Albeck S , Prilusky J , Lieberman RL , Aharoni A , Silman I , Sussman JL , Tawfik DS , Fleishman SJ
Ref : Mol Cell , 63 :337 , 2016
Abstract : Upon heterologous overexpression, many proteins misfold or aggregate, thus resulting in low functional yields. Human acetylcholinesterase (hAChE), an enzyme mediating synaptic transmission, is a typical case of a human protein that necessitates mammalian systems to obtain functional expression. We developed a computational strategy and designed an AChE variant bearing 51 mutations that improved core packing, surface polarity, and backbone rigidity. This variant expressed at approximately 2,000-fold higher levels in E. coli compared to wild-type hAChE and exhibited 20 degrees C higher thermostability with no change in enzymatic properties or in the active-site configuration as determined by crystallography. To demonstrate broad utility, we similarly designed four other human and bacterial proteins. Testing at most three designs per protein, we obtained enhanced stability and/or higher yields of soluble and active protein in E. coli. Our algorithm requires only a 3D structure and several dozen sequences of naturally occurring homologs, and is available at http://pross.weizmann.ac.il.
ESTHER : Goldenzweig_2016_Mol.Cell_63_337
PubMedSearch : Goldenzweig_2016_Mol.Cell_63_337
PubMedID: 27425410
Gene_locus related to this paper: human-ACHE

Title : A new post-intoxication treatment of paraoxon and parathion poisonings using an evolved PON1 variant and recombinant GOT1 - Goldsmith_2016_Chem.Biol.Interact_259_242
Author(s) : Goldsmith M , Ashani Y , Margalit R , Nyska A , Mirelman D , Tawfik DS
Ref : Chemico-Biological Interactions , 259 :242 , 2016
Abstract : Organophosphate (OP) based pesticides are highly toxic compounds that are still widely used in agriculture around the world. According to World Health Organization (WHO) data, it is estimated that between 250,000 and 370,000 deaths occur yearly around the globe as a result of acute intoxications by pesticides. Currently available antidotal drug treatments of severe OP intoxications are symptomatic, do not reduce the level of intoxicating OP in the body and have limited ability to prevent long-term brain damage. Pesticide poisonings present a special therapeutic challenge since in many cases, such as with parathion, their toxicity stems from their metabolites that inhibit the essential enzyme acetylcholinesterase. Our goal is to develop a new treatment strategy for parathion intoxication by combining a catalytic bioscavenger that rapidly degrades the intoxicating parathion-metabolite (paraoxon) in the blood, with a glutamate bioscavenger that reduces the elevated concentration of extracellular glutamate in the brain following OP intoxication. We report on the development of a novel catalytic bioscavenger by directed evolution of serum paraoxonase 1 (PON1) that effectively detoxifies paraoxon in-vivo. We also report preliminary results regarding the utilization of this PON1 variant together with a recombinant human enzyme glutamate oxaloacetate transaminase 1 (rGOT1), suggesting that a dual PON-GOT treatment may increase survival and recovery from parathion and paraoxon intoxications.
ESTHER : Goldsmith_2016_Chem.Biol.Interact_259_242
PubMedSearch : Goldsmith_2016_Chem.Biol.Interact_259_242
PubMedID: 27256520

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 : Enzyme engineering by targeted libraries - Goldsmith_2013_Methods.Enzymol_523_257
Author(s) : Goldsmith M , Tawfik DS
Ref : Methods Enzymol , 523 :257 , 2013
Abstract : This review outlines the strategies we apply for directed enzyme evolution using targeted libraries, namely, libraries that diversify specific residues with predefined mutational compositions. The theoretical grounds underlining the design of such libraries are described, including the mutational load, the ratio of beneficial versus deleterious mutations, and screening capacity. We point out the advantage of using mutational spiking strategies for "hedging the bets," exploring a large number of potentially beneficial mutations, and tuning the library's mutational load. Also highlighted are the merits of low-throughput screens that measure multiple parameters at high accuracy, and of using the desired substrate and reaction conditions rather than surrogates. We subsequently describe library construction strategies (rational and analytical) based on structure and sequence analyses, including ancestral libraries, which are particularly suitable for low-throughput screens. We also discuss the critical role of including compensatory, stabilizing mutations during library construction. Finally, the design efficiency and the optimal mutational loads of libraries are assessed by comparing targeted mutational libraries versus libraries of random mutations.
ESTHER : Goldsmith_2013_Methods.Enzymol_523_257
PubMedSearch : Goldsmith_2013_Methods.Enzymol_523_257
PubMedID: 23422434

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 : Directed enzyme evolution: beyond the low-hanging fruit - Goldsmith_2012_Curr.Opin.Struct.Biol_22_406
Author(s) : Goldsmith M , Tawfik DS
Ref : Current Opinion in Structural Biology , 22 :406 , 2012
Abstract : The field of directed evolution has progressed to the point where it is feasible to engineer enzymes for unnatural substrates and reactions with catalytic efficiencies and regio-specificity or stereo-specificity that rival those of natural enzymes. Here, we describe the conceptual and methodological advances that have enabled this progress. We address methodologies based on small libraries enriched with improved variants and carrying compensatory stabilizing mutations. Such libraries can be combined with low-throughput screens that provide high accuracy and directly target the desired substrate and reaction conditions, and thereby provide highly improved variants.
ESTHER : Goldsmith_2012_Curr.Opin.Struct.Biol_22_406
PubMedSearch : Goldsmith_2012_Curr.Opin.Struct.Biol_22_406
PubMedID: 22579412

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 : 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 : Potential role of phenotypic mutations in the evolution of protein expression and stability - Goldsmith_2009_Proc.Natl.Acad.Sci.U.S.A_106_6197
Author(s) : Goldsmith M , Tawfik DS
Ref : Proc Natl Acad Sci U S A , 106 :6197 , 2009
Abstract : Phenotypic mutations (errors occurring during protein synthesis) are orders of magnitude more frequent than genetic mutations. Consequently, the sequences of individual protein molecules transcribed and translated from the same gene can differ. To test the effects of such mutations, we established a bacterial system in which an antibiotic resistance gene (TEM-1 beta-lactamase) was transcribed by either a high-fidelity RNA polymerase or its error-prone mutant. This setup enabled the analysis of individual mRNA transcripts that were synthesized under normal or error-prone conditions. We found that an increase of approximately 20-fold in the frequency of transcription errors promoted the evolution of higher TEM-1 expression levels and of more stable enzyme variants. The stabilized variants exhibited a distinct advantage under error-prone transcription, although under normal transcription they conferred resistance similar to wild-type TEM-1. They did so, primarily, by increasing TEM-1's tolerance to destabilizing deleterious mutations that arise from transcriptional errors. The stabilized TEM-1 variants also showed increased tolerance to genetic mutations. Thus, although phenotypic mutations are not individually subjected to inheritance and natural selection, as are genetic mutations, they collectively exert a direct and immediate effect on protein fitness. They may therefore play a role in shaping protein traits such as expression levels, stability, and tolerance to genetic mutations.
ESTHER : Goldsmith_2009_Proc.Natl.Acad.Sci.U.S.A_106_6197
PubMedSearch : Goldsmith_2009_Proc.Natl.Acad.Sci.U.S.A_106_6197
PubMedID: 19339491