WO2011033506A2

WO2011033506A2 - Isolated pon1 polypeptides, polynucleotides encoding same and uses thereof in treating or preventing organophosphate exposure associated damage

Info

Publication number: WO2011033506A2
Application number: PCT/IL2010/000754
Authority: WO
Inventors: Dan S. Tawfik; Rinkoo Devi Gupta; Moshe Goldsmith; Yaacov Ashani; Moshe Ben-David; Israel Silman; Joel L. Sussman; Haim Leader
Original assignee: Yeda Research And Development Co. Ltd.
Priority date: 2009-09-17
Filing date: 2010-09-15
Publication date: 2011-03-24
Also published as: EP2478093A2; WO2011033506A9; WO2011033506A3

Abstract

An isolated polypeptide comprising the amino acid sequence of serum paraoxonase (PON1) having catalytic efficiency of k _cat/K_M ≈10⁶ M^-1min^-1 for a nerve-agent substrate.

Description

ISOLATED PON1 POLYPEPTIDES, POLYNUCLEOTIDES ENCODING SAME AND USES THEREOF IN TREATING OR PREVENTING ORGANOPHOSPHATE

EXPOSURE ASSOCIATED DAMAGE RELATED APPLICATION/S

This application claims the benefit of priority under 35 USC 119(e) of U.S. Provisional Patent Application No. 61/272,363 filed September 17, 2009, the contents of which are incorporated herein by reference in their entirety.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to isolated PON1 polypeptides, polynucleotides encoding same and uses thereof in treating or preventing organophosphate exposure associated damage.

Inhibitors of acetylcholinesterase (AChE), including organophosphate (OP)-based pesticides and nerve agents, threaten both military and civilian populations. A timely pharmacological treatment with atropine and oxime AChE reactivators can save lives but in many cases does not prevent cholinergic crisis and the resulting onset of secondary toxic manifestations induced by OP intoxication. Side effects associated with drugs such as pyridostigmine used as protective treatment prior to OP exposure have also prompted the search for effective prophylactics and antidotes. Rather than minimizing the damages caused by the OP, the goal of prophylactic drugs is to intercept the OPs before they even reach their target organs. A stoicheiometric bioscavenger based on human butyrylcholinesterase has been recently developed. However, owing to the daunting mass ratio of OP to protein, hundreds of mgs of protein are required to confer protection against exposure to doses beyond a single LD50 dose [Ashani, Y. & Pistinner, S. Estimation of the upper limit of human butyrylcholinesterase dose required for protection against organophosphates toxicity: a mathematically based toxicokinetic model. Toxicol Sci 77, 358-67 (2004)]. Catalytic scavengers, namely enzymes displaying multiple turnovers, may allow rapid and efficient protection against high OP doses using low protein amounts [Ditargiani, R. C, Chandrasekaran, L., Belinskaya, T. & Saxena, A. In search of a catalytic bioscavenger for the prophylaxis of nerve agent toxicity. Chem Biol Interact [Epub ahead of print] (2010]. However, with few exceptions, xenobiotics such as OPs are promiscuous substrates for natural enzymes and are degraded with low catalytic efficiencies. Improved OP hydrolyzing enzyme variants have been engineered (e.g. PTE, DFPase, Hill, C. M., Li, W. S., Thoden, J. B., Holden, H. M. & Raushel, F. M. Enhanced degradation of chemical warfare agents through molecular engineering of the phosphotriesterase active site. J Am Chem Soc 125, 8990- 1 (2003), Mee-Hie Cho, C, Mulchandani, A. & Chen, W. Functional analysis of organophosphorus hydrolase variants with high degradation activity towards organophosphate pesticides. Protein Eng Des Sel 19, 99-105 (2006), Melzer, M. et al. Reversed enantioselectivity of diisopropyl fluorophosphatase against organophosphorus nerve agents by rational design. J Am Chem Soc 131, 17226-32 (2009)), but prophylactic protection from >lXLD₅o doses at reasonable protein amounts requires catalytic scavengers whose efficiencies in k_cat/KM terms are >10⁷ M^-1 mm^-1.

The G-agents cyclosarin (GF) and soman (GD) comprise a prime target for, scavenger-based prophylaxis due to the low efficacy of pharmacological drugs used to counteract their toxicity [Kassa, J., Karasova, J. Z., Caisberger, F. & Bajgar, J. The influence of combinations of oximes on the reactivating and therapeutic efficacy of antidotal treatment of soman poisoning in rats and mice. Toxicol Mech Methods 19, 547-51 (2009)]. Although applied as racemates, their S_p isomers comprise the tangible threat (Figure 5). Unfortunately, enzymes tested thus far primarily hydrolyze less toxic R_p isomer [Harvey, S. P. et al. Stereospecificity in the enzymatic hydrolysis of cyclosarin (GF). Enzyme and Microbial Technology 37, 547-555 (2005); Li, W. S., Lum, K. T., Chen-Goodspeed, M., Sogorb, M. A. & Raushel, F. M. Stereoselective detoxification of chiral sarin and soman analogues by phosphotriesterase. Bioorg Med Chem 9, 2083-91 (2001)].

Additional background art includes:

WO2004/078991 SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present invention there is provided an isolated polypeptide comprising an amino acid sequence of serum paraoxonase (PON1) having catalytic efficiency of k_catIKM ~10⁶ M^-1min^-1 for a nerve- agent substrate.

According to some embodiments of the invention, the nerve-agent substrate comprises an Sp isomer. According to some embodiments of the invention, the isolated polypeptide has catalytic efficiency of k_catIKM ~10⁷ M^min^-1 for Sp nerve-agent substrates.

According to some embodiments of the invention, the amino acid sequence of serum paraoxonase (PON1) comprises a mutation selected from the group consisting of: L69G/A/L/V/S/M, K70A/S/Q/N, Y71/F/C/A/L/I, H115W/L/V/C, H134R/N, F222S/M/C, F292S/V/L, T332S/M/C/A, M196V/L/F, V97A ,V346A ,N41D, Y293S, V97A, V276A , T326S, SUIT, SHOP, P135A, N41D, N324D, M289I, L240S/V, L14M, L10S, K233E, H285R, H243R,, F28Y, F264L, D309N/G, A6E, N227L, F178V,D49N, wherein the amino acid coordinates correspond to the G3C9 PON1 variant.

According to some embodiments of the invention, the isolated polypeptide is expressible in bacteria.

According to some embodiments of the invention, the amino acid sequence is selected from the group consisting of the sequences set forth in SEQ ID NO: 129, 2-54 and 120-128.

According to some embodiments of the invention, the amino acid sequence is selected from the group consisting of the sequences set forth in SEQ ID NO: 129, 2, 4, 7, 9, 12, 24, 47, 53, 120-128.

According to an aspect of some embodiments of the present invention there is provided an isolated polynucleotide comprising a nucleic acid sequence encoding the polypeptide.

According to an aspect of some embodiments of the present invention there is provided a pharmaceutical composition comprising as an active ingredient the isolated polypeptide and a pharmaceutically acceptable carrier.

According to an aspect of some embodiments of the present invention there is provided a nucleic acid construct comprising the isolated polynucleotide and a cis- regulatory element driving expression of the polynucleotide.

According to an aspect of some embodiments of the present invention there is provided a method of treating or preventing organophosphate exposure associated damage in a subject in need thereof, the method comprising providing the subject with a therapeutically effective amount of the isolated polypeptide to thereby treat the organophosphate exposure associated damage in the subject. According to some embodiments of the invention, the providing is effected prior to the organophosphate exposure.

According to some embodiments of the invention, the providing is effected by inhalation administration.

According to some embodiments of the invention, the providing is effected 10 hours prior to the exposure until 7 days following exposure.

According to some embodiments of the invention, the providing is effected by inhalation and injection.

According to some embodiments of the invention, the method further comprises administering to the subject atropine and optionally oxime.

According to some embodiments of the invention, the providing is effected by topical application.

According to an aspect of some embodiments of the present invention there is provided an article of manufacture for treating or preventing organophosphate exposure associated damage, the article of manufacture comprising the isolated polypeptide immobilized on to a solid support.

According to some embodiments of the invention, the solid support is for topical administration.

According to some embodiments of the invention, the solid support for topical administration is selected from the group consisting of a sponge, a wipe and a fabric.

According to some embodiments of the invention, the solid support is selected from the group consisting of a filter, a fabric and a lining.

According to an aspect of some embodiments of the present invention there is provided a method of detoxifying a surface, the method comprising contacting the surface with the isolated polypeptide, thereby detoxifying the surface.

According to some embodiments of the invention, the method further comprises contacting the surface with a decontaminating foam, a combination of baking condition heat and carbon dioxide, or a combination thereof.

According to some embodiments of the invention, the polypeptide is comprised in a coating, a paint, a non-film forming coating, an elastomer, an adhesive, an sealant, a material applied to a textile, or a wax. Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

In the drawings:

FIG. 1 is a scheme illustrating the pET32PONl plasmid. This plasmid was used for the expression of PON1 variants with a C-terminal His-tag and no GFP. The plasmid was derived from pET32b(+) from which the thioredoxin fusion protein and peptide tags were truncated using the Notl/Xhol sites. The recombinant PON1 variant G3C9, and library variants, were inserted using the NcoIINotI sites. The Notl restriction site was inserted upstream to the His tag to enable the cloning of various PON1 variants with no alterations to the tag.

FIGs. 2A-C are graphs of FACS detection and sorting of PONl-carrying E. coli cells in w/o/w emulsion droplets. E. coli BL21 (DE3) cells possessing GFPuv gene in the genome were used for expression of the PON 1 under the T7 promoter. Cells were emulsified, together with the fluorogenic substrate (DEPCyC). Briefly, filtered cells were compartmentalized in the first emulsion (water-in-oil), and 100 mM solutions of DEPCyC was added to the oil phase (0.8 μl , to a final concentration of 50μΜ).

The production of the second emulsion (water-in-oil-in-water) and sorting were performed as described⁴. More than 10⁶ events, at 2000 events/sec, were sorted using FACSA ia (Becton-Dickinson). Events corresponding to single E. coli cells were gated by GFP emission (at 530nm, using blue laser for excitation). Figure 2A - Representative density plot FSC-H (forward scatter) and SSC-H (side scatter) analysis of the double emulsion. Figure 2B - Histogram of the GFP emission for the Rl population of droplets. Events gated in R2 correspond to droplets that contain GFP expressing cells. Figure 2C - The R1+R2 gated events were analyzed for the hydrolytic activity. Events gated in R3 represent active variants that were present as 0.5-1 % of total population; these were sorted into liquid growth media.

FIG. 3 is a graph illustrating kinetic parameters. Shown is a representative Michaelis-Menten plot for rePON1 variants 8C8, 0C9, and 3D8, evolved towards Sp- CMP-MeCyC hydrolysis. Enzyme concentrations were 0.65 μΜ for 8C8, and 12.5 nM for 0C9 and 3D8. Substrate concentrations were varied from 0.4 μΜ up to 1000 μΜ.;

FIG. 4 is a graph showing the effect of excess of free coumarin on the hydrolysis of CMP-F by variant 4E9. The kinetics of CMP-F (40 nM) hydrolysis by 4E9 (16 nM) were determined with and without the addition of a 4-fold excess of free coumarin (64nM).

FIG. 5 shows some organophosphates (Ops) used herein. Shown are the two enatiomers of G-agents: cyclosarin (GF, R= cyclohexyl), sarin (GB, R= iso-propyl) and soman (GD, R= pinacolyl). For consistency, the fluorogenic analogues (X= 3-cyano-7- hydroxy-4-methylcoumarin) are dubbed CMP-coumarin, IMP- coumarin, and Pin- coumarin, respectively, and the actual agents (X=F) CMP-F, IMP-F, and Pin-F, respectively.

FIGs. 6A-C shows the hydrolysis of CMP-coumarin and CMP-F by rePONl variants. Enzyme concentrations were varied depending on the variant's activity, and are noted in the figure. Figure 6A. Hydrolysis of racemic CMP-coumarin (12μΜ) in the presence of variants 4E9, 3D8, 3B3 (plus addition of 0.03 μΜ 4E9 after 6 mins; indicated by the black arrow), and wild-type-like rePONl (plus addition of 0.03 μΜ 4E9 after 20 mins). Figure 6B. Hydrolysis of Sp-CMP-coumarin (6μΜ) in the presence of variants 4E9, 3D8, 3B3, and rePONl. Figure 6C. Residual AChE activity was assayed following the incubation of in-situ generated CMP-F (40nM) and 4E9, 3D8, 3B3, and rePONl; the data were fitted to a first-order rate equation to derive the apparent rate constant for hydrolysis of CMP-F. DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

Organophosphates (OPs), including pesticides and nerve agents, comprise a prime target for detoxification. Albeit, no natural enzymes are available that proficiently degrade most of these xenobiotics. Obtaining highly proficient OP hydrolases, and in particular for the more toxic stereoisomer Sp of the G-type nerve agents remains a challenge.

The present inventors generated through laborious experimentation and screening a series of variants of mammalian serum paraoxonase (PON1) - an enzyme that is potentially applicable in vivo, with sufficiently high catalytic efficiency for detoxification (k_cat/K_m≥10⁷ M^-1min^-1). Directed evolution of PON1 using structure- based as well as random mutagenesis, and combining low-throughput methodologies (96-well plate screening) with high-throughput screens using compartmentalization in emulsions, enabled taking wild-type-like PON1 that has no detectable activity with Sp G-type OPs, and generating variants with catalytic efficiency of >10⁷ M^-1min^-1 While the directed evolution used model OPs with a fluorogenic leaving group, a final screen was done using an acetylcholinesterase inhibition assay and in-situ generated nerve agents to identify highly proficient variants that can hydrolyse the actual nerve agents.

The present detoxification model was also validated by demonstrating prophylactic protection in an animal model. The differences in survival and intoxication symptoms between mice pretreated with the evolved variant 4E9 and mice pretreated with the conventional atropine-oxime treatment probably relate to the very different effects of these treatments - atropine plus 2-PAM aims to minimize the damages of the OP, whereas rePON-4E9 neutralizes the agent before it even reaches its target. In conclusion, there is a direct correlation between the catalytic efficiency of evolved PONl variants at OP hydrolysis in-vitro and the ability of these variants to act as effective prophylactics in-vivo.

The newly isolated rePONl variants, and the methodologies described here, also provide the basis for further engineering of PONl towards other G-type nerve agents, e.g. sarin, and soman. The evolved variants hydrolyze these agents, and soman (GD) in particular, at relatively high rates (4E9's apparent k_cat/KM value for sarin (IMP-F) is <3xl0⁵ M^min ¹, and for soman (Pin-F), 7.4xl0⁶ M^-1min^-1, and 0.58xl0⁶ M^min^-1, for the two toxic isomers respectively). 1-I-Fll exhibits k_cat/ =4*10⁶ (M^rnin ¹) with the toxic isomer of Sarin (GB) and a catalytic rate of k_cat/ M=4.4*10⁷ (M^-1min ¹) for all isomers of Soman (GD). A catalytic efficiency of k_cat/K =4.6*10⁷ (M^min^-1) for the more toxic isomer of GF is exhibited with the 1-I-Fll enzyme.

Thus, according to an aspect of the invention there is provided an isolated polypeptide comprising an amino acid sequence of serum paraoxonase (PONl) having catalytic efficiency of k_cat/KM ~10⁶ M^-1min^-1 for a nerve-agent substrate.

As used herein the term "serum paraoxonase (PONl)" refers to a naturally occurring or man-made sequence. PONl (EC 3.1.8.1 or EC 3.1.1.2 e.g., PON1_HUMAN, P27169) is a high-density lipoprotein (HDL)-associated serum enzyme whose primary physiological role is to protect low-density lipoproteins (LDLs) from oxidative modifications. PONl can also hydrolyze organophosphorus (OP) compounds, including commonly used insecticides, and its name derives from one of its most commonly used in vitro substrates— paraoxon. More recently, in addition to its role in lipid metabolism and, hence, in cardiovascular disease and arteriosclerosis, PONl has also been shown to be involved in the metabolism of lactones and cyclic carbonates. Early studies of enzymatic activity in serum indicated a bimodal or trimodal distribution in Caucasian populations. Two main polymorphisms in the coding region, as well as five in the 5' regulating region, have been characterized. The Q192R polymorphism determines the catalytic efficiency of hydrolysis of some substrates, and certain promoter polymorphisms, in particular C-108T, contribute to the level of expression of PONl. Recently, additional polymorphisms in the coding region, 5' regulatory region, and PONl introns have been reported.

Any PONl may be used e.g., human PONl, rabbit PONl. Others are listed below (Table lal).

In a specific embodiment the enzyme is expressible in E. Coli such as the PON1 variant G3C9 having GenBank Accession AY499193 (see e.g., WO2004/078991, which describes this variant and other equivalent variants and is hereby incorporated by reference in its entirety).

As used herein, a "nerve agent" refers to an organophosphate (OP) compound such as having an acetylcholinesterase inhibitory activity. The toxicity of an OP compound depends on the rate of its inhibition of acetylcholinesterase with the concomitant release of the leaving group such as fluoride, alkylthiolate, cyanide or aryoxy group. The nerve agent may be a racemic composition or a purified enantiomer (e.g., Sp or Rp).

According to a specific embodiment, the nerve agent substrate comprises an Sp isomer.

Certain OP compounds are so toxic to humans that they have been adapted for use as chemical warfare agents (CWAs), such as tabun, soman, sarin, cyclosarin, VX, and R-VX. A CWA may be in airborne form and such a formulation is known herein as an "OP-nerve gas." Examples of airborne forms include a gas, a vapor, an aerosol, a dust, or a combination thereof. Examples of an OP compounds that may be formulated as an OP nerve gas include tabun, sarin, soman, cyclosarin, VX, GX or a combination thereof. An example of an organophosphate which is close to, albeit not similar in its properties to those of the nerve gases is that of DFP, diisopropylfluorophosphonate, which is considerably less volatile than certain members of this group. In addition to the initial inhalation route of exposure common to such agents, CWAs, especially persistent agents such as VX and thickened soman, pose threats through dermal absorption [In "Chemical Warfare Agents: Toxicity at Low Levels," (Satu M. Somani and James A. Romano, Jr., Eds.) p. 414, 2001]. Such persistent CWA agents remain as a solid or liquid while exposed to the open air for more than three hours. Often after release, a persistent agent may convert from an airborne dispersal form to a solid or liquid residue on a surface, thus providing the opportunity to contact the skin of a human.

Examples of an OP pesticide include bromophos-ethyl, chlorpyrifos, chlorfenvinphos, chlorothiophos, chlorpyrifos-methyl, coumaphos, crotoxyphos, crufomate, cyanophos, diazinon, dichlofenthion, dichlorvos, dursban, EPN, ethoprop, ethyl-parathion, etrimifos, famphur, fensulfothion, fenthion, fenthrothion, isofenphos, jodfenphos, leptophos-oxon, malathion, methyl-parathion, mevinphos, paraoxon, parathion, parafhion-methyl, pirimiphos-ethyl, pirimiphos-methyl, pyrazophos, quinalphos, ronnel, sulfopros, sulfotepp, trichloronate, or a combination thereof.

Methods of selecting PON1 polypeptides with the desired activity are provided in the Examples section below. Typically, these methods involve directed evolution of PON1 using structure-based as well as random mutagenesis, and combining low- throughput methodologies (96-well plate screening) with high-throughput screens e.g., using compartmentalization in emulsions,

As used herein the phrase "in vitro evolution process" (also referred to as "a directed evolution process") refers to the manipulation of genes and selection or screening of a desired activity. A number of methods, which can be uilized to effect in vitro evolution, are known in the art. One approach of executing the in-vitro evolution process is provided in the Examples section.

General outline of directed evolution is provided in Tracewell CA, Arnold FH "Directed enzyme evolution: climbing fitness peaks one amino acid at a time" Curr Opin Chem Biol. 2009 Feb;13(l):3-9. Epub 2009 Feb 25; Gerlt JA, Babbitt PC, Curr Opin Chem Biol. 2009 Feb;13(l):10-8. Epub 2009 Feb 23 and WO2004/078991 (either of which is hereby incorporated by reference in its entirety).

Methods of producing recombinant proteins are well known in the art. According to a specific embodiment, mutations which may be employed to improve the hydrolytic efficiency of PON1 to nerve agent substrates comprise mutations in at least one of the following residues, F28, N41, E53, D54, L69, K70, Y71, P72, G73, 174, M75, H115, G116, H134, V167, N168, D169, T181, D183, H184, M196, F222, A223, N224, G225, L240, L241, L267, V268, D269, N270, C284, H285, N287, G288, R290, 1291, F292, F293, Y294, G330, S331, T332, V346, F347 V436 Y293, V276, T326, Sill, S110, P135, N41, N324, M289, L240, L14, L10, K233, H285, H243, F28, F264, D309, A6, N227, F178 and D49, where the coordinates corresponds to the PON1 variant G3C9 (SEQ ID NO: 1) having GenBank Accession AY499193. Amino acid coordinates should be adapted easily to PON1 variants of the same or other species by amino acid sequence alignments which may be done manually or using specific bioinformatic tools such as FASTA. L-ALIGN and protein Blast,

and the catalytic Ca.

Some exemplary mutations include but are not limited to L69G/A/L/V/S/M, K70A/S/Q/N, Y71/F/C/A/L/I, H115W/L/V/C, H134R/N, F222S/M/C, F292S/V/L, T332S/M/C/A, M196V/L/F, V97A ,V346A ,N41D, Y293S, V276A, T326S, SUIT, SHOP, P135A, N41D, N324D, M289I, L240S/V, L14M, L10S, K233E, H285R, H243R, F28Y, F264L, D309N/G, A6E, N227L, F178V,D49N.

Thus the present teachings provide for an isolated polypeptide comprising an amino acid sequence of serum paraoxonase (PON1) having catalytic efficiency between the range of k_cMIK_M ~10⁶ -10⁸ M^min^-1, or specifically of k_caC/K_M ~10⁶ M^-1 min , kcatlK_M ~5x10⁶ M^-1W^-1, k_cat/K_M ~10⁷ M^min^-1, k_cat/K_M ~5xl 0⁷ M^min^"1, k_catIK_M =10⁸ M^min ¹ for nerve-agent substrates (e.g., Sp isomers).

The polypeptides of the present invention are preferably expressible in bacteria such as E.coli [e.g., BL21, BL21 (DE3), Origami B (DE3), available from Novagen (www.calbiochem.com ) and RIL (DE3) available from Stratagene, (www.stratagene.com). Essentially, at least 2 %, at least 5 %, at least 10 %, at least 20 %, at least 30 %, at least 40 %, at least 50 %, at least 60 %, at least 70 %, at least 80 %, at least 90 %, at least 95 % or more, say 100 %, of bacterially expressed protein remains soluble (i.e., does not precipitate into inclusion bodies).

According to some embodiments of the invention, the amino acid sequence of the polypeptide is selected from the group consisting of the sequences set forth in SEQ ID NO: 129, 2-54 and 120-128. According to a specific embodiment, the isolated polypeptide is selected from the list below (Table la2). Other polypeptides are listed in the Examples section which follows.

As used herein the term "isolated" refers to isolated from the natural environment e.g., serum.

The term "polypeptide" as used herein encompasses native polypeptides (synthetically synthesized polypeptides or recombinant polypeptides) and peptidomimetics, as well as peptoids and semipeptoids which are peptide analogs, which may have, for example, modifications rendering the polypeptides more stable while in a body or more capable of penetrating into cells. Such modifications include, but are not limited to N terminus modification, C terminus modification, peptide bond modification, including, but not limited to, CH2- H, CH2-S, CH2-S=0, 0=C-NH, CH2-0, CH2-CH2, S=C-NH, CH=CH or CF=CH, backbone modifications, and residue modification. Methods for preparing peptidomimetic compounds are well known in the art and are specified, for example, in Quantitative Drug Design, C.A. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992), which is incorporated by reference as if fully set forth herein. Further details in this respect are provided hereinunder.

Peptide bonds (-CO-NH-) within the peptide may be substituted, for example, by

N-methylated bonds (-N(CH3)-CO-), ester bonds (-C(R)H-C-0-0-C(R)-N-), ketomethylen bonds (-CO-CH2-), a-aza bonds (-NH-N(R)-CO-), wherein R is any alkyl, e.g., methyl, carba bonds (-CH2-NH-), hydroxyethylene bonds (-CH(OH)-CH2-), thioamide bonds (-CS-NH-), olefinic double bonds (-CH=CH-), retro amide bonds (- NH-CO-), peptide derivatives (-N(R)-CH2-CO-), wherein R is the "normal" side chain, naturally presented on the carbon atom.

Synthetic amino acid substitutions may be employed to improve stability and bioavailability.

Table la3 below lists non-conventional or modified amino acids e.g., synthetic, which can be used with the present invention.

The present teachings also provide for nucleic acid sequences encoding such PON1 polypeptides.

Thus, according to an aspect of the present invention there is provided an isolated polynucleotide including a nucleic acid sequence, which encodes the isolated polypeptide of the present invention. As used herein the phrase "an isolated polynucleotide" refers to a single or a double stranded nucleic acid sequence which is isolated and provided in the form of an RNA sequence, a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence and/or a composite polynucleotide sequences (e.g., a combination of the above).

As used herein the phrase "complementary polynucleotide sequence" refers to a sequence, which results from reverse transcription of messenger RNA using a reverse transcriptase or any other RNA dependent DNA polymerase. Such a sequence can be subsequently amplified in vivo or in vitro using a DNA dependent DNA polymerase.

As used herein the phrase "genomic polynucleotide sequence" refers to a sequence derived (isolated) from a chromosome and thus it represents a contiguous portion of a chromosome.

As used herein the phrase "composite polynucleotide sequence" refers to a sequence, which is at least partially complementary and at least partially genomic. A composite sequence can include some exonal sequences required to encode the polypeptide of the present invention, as well as some intronic sequences interposing therebetween. The intronic sequences can be of any source, including of other genes, and typically will include conserved splicing signal sequences. Such intronic sequences may further include cis acting expression regulatory elements.

According to an exemplary embodiment the polynucleotide is selected from the group consisting of 56-108 and 130-139.

Polypeptides of the present invention can be synthesized using recombinant DNA technology or solid phase technology.

Recombinant techniques are preferably used to generate the polypeptides of the present invention. Such recombinant techniques are described by Bitter et al., (1987) Methods in Enzymol. 153:516-544, Studier et al. (1990) Methods in Enzymol. 185:60- 89, Brisson et al. (1984) Nature 310:511-514, Takamatsu et al. (1987) EMBO J. 6:307- 311, Coruzzi et al. (1984) EMBO J. 3:1671-1680 and Brogli et al., (1984) Science 224:838-843, Gurley et al. (1986) Mol. Cell. Biol. 6:559-565 and Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421-463. To produce a polypeptide of the present invention using recombinant technology, a polynucleotide encoding a polypeptide of the present invention is ligated into a nucleic acid expression construct, which includes the polynucleotide sequence under the transcriptional control of a cis-regulatory (e.g., promoter) sequence suitable for directing constitutive or inducible transcription in the host cells, as further described hereinbelow.

Other than containing the necessary elements for the transcription and translation of the inserted coding sequence, the expression construct of the present invention can also include sequences (i.e., tags) engineered to enhance stability, production, purification, yield or toxicity of the expressed polypeptide. Such a fusion protein can be designed so that the fusion protein can be readily isolated by affinity chromatography; e.g., by immobilization on a column specific for the heterologous protein. Where a cleavage site is engineered between the peptide moiety and the heterologous protein, the peptide can be released from the chromatographic column by treatment with an appropriate enzyme or agent that disrupts the cleavage site [e.g., see Booth et al. (1988) Immunol. Lett. 19:65-70; and Gardella et al, (1990) J. Biol. Chem. 265:15854-15859].

A variety of prokaryotic or eukaryotic cells can be used as host-expression systems to express the polypeptide coding sequence. These include, but are not limited to, microorganisms, such as bacteria transformed with a recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vector containing the polypeptide coding sequence; yeast transformed with recombinant yeast expression vectors containing the polypeptide coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors, such as Ti plasmid, containing the polypeptide coding sequence. Mammalian expression systems can also be used to express the polypeptides of the present invention. Bacterial systems are preferably used to produce recombinant polypeptides, according to the present invention, thereby enabling a high production volume at low cost.

Other expression systems such as insects and mammalian host cell systems, which are well known in the art can also be used by the present invention. In any case, transformed cells are cultured under effective conditions, which allow for the expression of high amounts of recombinant polypeptides. Effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that permit protein production. An effective medium refers to any medium in which a cell is cultured to produce the recombinant polypeptides of the present invention. Such a medium typically includes an aqueous solution having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutrients, such as vitamins. Cells of the present invention can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes, and petri plates. Culturing can be carried out at a temperature, pH and oxygen content appropriate for a recombinant cell. Such culturing conditions are within the expertise of one of ordinary skill in the art.

Depending on the vector and host system used for production, resultant proteins of the present invention may either remain within the recombinant cell; be secreted into the fermentation medium; be secreted into a space between two cellular membranes, such as the periplasmic space in E. coli; or be retained on the outer surface of a cell or viral membrane.

Following a certain time in culture, recovery of the recombinant protein is effected. The phrase "recovering the recombinant protein" refers to collecting the whole fermentation medium containing the protein and need not imply additional steps of separation or purification. Proteins of the present invention can be purified using a variety of standard protein purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential solubilization.

Polypeptides of the present invention can be used for treating an organophosphate exposure associated damage.

Thus according to an aspect of the invention there is provided a method of treating or preventing organophosphate exposure associated damage in a subject in need thereof, the method comprising providing the subject with a therapeutically effective amount of the isolated polypeptide described above to thereby treat the organophosphate exposure associated damage in the subject.

As used herein the term "treating" refers to preventing, curing, reversing, attenuating, alleviating, minimizing, suppressing or halting the deleterious effects of the immediate life-threatening effects of organophosphate intoxication and its long-term debilitating consequences.

As used herein the phrase "organophosphate exposure associated damage" refers to short term (e.g., minutes to several hours post- exposure) and long term damage (e.g., one week up to several years post- exposure) to physiological function (e.g., motor and cognitive functions). Organophosphate exposure associated damage may be manifested by the following clinical symptoms including, but not limited to, headache, diffuse muscle cramping, weakness, excessive secretions, nausea, vomiting and diarrhea. The condition may progress to seizure, coma, paralysis, respiratory failure, delayed neuropathy, muscle weakness, tremor, convulsions, permanent brain dismorphology, social/behavioral deficits and general cholinergic crisis (which may be manifested for instance by exacerbated inflammation and low blood count. Extreme cases may lead to death of the poisoned subjects.

As used herein the term "organophosphate compound" refers to a compound comprising a phosphoryl center, and further comprises two or three ester linkages. In some aspects, the type of phosphoester bond and/or additional covalent bond at the phosphoryl center classifies an organophosphorus compound. In embodiments wherein the phosphorus is linked to an oxygen by a double bond (PdbdO), the OP compound is known as an "oxon OP compound" or "oxon organophosphorus compound." In embodiments wherein the phosphorus is linked to a sulfur by a double bond (PdbdS), the OP compound is known as a "thion OP compound" or "thion organophosphorus compound."

Additional examples of bond-type classified OP compounds include a phosphonocyanidate, which comprises a P--CN bond; a phosphoroamidate, which comprises a P--N bond; a phosphotriester, which comprises a P(— 0-Rl)₃ bond; a phosphodiester, which comprises a P(--0-R)2 bond, where R is alkyl or aryl moieties; a phosphonofluoridate, which comprises a P--F bond; and a phosphonothiolate, which comprises a P— S-alkyl or P— S-alkyl-N(R')₂ bond, where R is any alkyl group. A "dimethoxy OP compound" comprises two methyl moieties covalently bonded to the phosphorus atom, such as, for example, malathion. A "diethyl OP compound" comprises two ethoxy moieties covalently bonded to the phosphorus atom, such as, for example, diazinon or paraoxon.

In general embodiments, an OP compound comprises an organophosphorus nerve agent or an organophosphorus pesticide.

As used herein the phrase "a subject in need thereof" refers to a human or animal subject who is sensitive to OP toxic effects. Thus, the subject may be exposed or at a risk of exposure- to OP. Examples include civilians contaminated by a terrorist attack at a public event, accidental spills in industry and during transportation, field workers subjected to pesticide/insecticide OP poisoning, truckers who transport pesticides, pesticide manufacturers, dog groomers who are overexposed to flea dip, pest control workers and various domestic and custodial workers who use these compounds, military personnel exposed to nerve gases.

As mentioned, in some embodiments of the invention the method is effected by providing the subject with a therapeutically effective amount of the PONl polypeptide of the invention.

As OP can be rapidly absorbed from lungs, skin, gastro-intestinal (GI) tract and mucous membranes, PONl may be provided by various administration routes or direct application on the skin.

For example, PONl may be immobilized on a solid support e.g., a porous support which may be a flexible sponge-like substance or like material, wherein the PONl is secured by immobilization. The support may be formed into various shapes, sizes and densities, depending on need and the shape of the mold. For example, the porous support may be formed into a typical household sponge, wipe or a towelette.

For example, such articles may be used to clean and decontaminate wounds, while the immobilized PONl will not leach into a wound. Therefore, the sponges can be used to decontaminate civilians contaminated by a terrorist attack at a public event.

Alternatively or additionally, PONl may be administered to the subject per se or in a pharmaceutical composition where it is mixed with suitable carriers or excipients.

As used herein a "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

Herein the term "active ingredient" refers to the PON1 accountable for the biological effect.

Hereinafter, the phrases "physiologically acceptable carrier" and

"pharmaceutically acceptable carrier" which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases.

Herein the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient.

Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

Techniques for formulation and administration of drugs may be found in

"Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference.

Suitable routes of administration may, for example, include oral, rectal, dermal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intravenous, inrtaperitoneal, intranasal, intrabone or intraocular injections.

Alternately, one may administer the pharmaceutical composition in a local rather than systemic manner, for example, via injection of the pharmaceutical composition directly into a tissue region (e.g., skin) of a patient. Topical administration is also contemplated according to the present teachings.

Pharmaceutical compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

For oral administration, the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

Pharmaceutical compositions which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.

For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

The pharmaceutical composition described herein may be formulated for parenteral administration, e.g., by bolus injection or continuos infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.

Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use. The pharmaceutical composition of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.

Pharmaceutical compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients (nucleic acid construct) effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., ischemia) or prolong the survival of the subject being treated.

Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays. For example, a dose can be formulated in animal models to achieve a desired concentration or titer (see the Examples section which follows). Such information can be used to more accurately determine useful doses in humans.

Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.l).

Dosage amount and interval may be adjusted individually to provide plasma or brain levels of the active ingredient are sufficient to induce or suppress the biological effect (minimal effective concentration, MEC). The MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.

Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.

The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.

PON1 may be administered prior to the OP exposure (prophylactically, e.g., 10 or 8 hours before exposure), and alternatively or additionally administered post exposure, even days after (e.g., 7 days) in a single or multiple-doses.

Embodiments of the invention also contemplate the use of other agents in combination with PON-1 for the treatment or prevention of OP damage. The following regimen is intended to encompass treatment with PON1 alone or in combination with other agents.

Thus, according to an exemplary embodiment, PON1 may be administered by inhalation to protect the lungs and injection (i.v.) to protect the circulation up to 2 hours post exposure. Atropine may be added 2-4 hours post exposure. Daily injections of PON1 may be administered up to 7 days post poisoning. Oximes like Hl-6 and mono- bisquaternary oximes such as pralidoxime chloride (2-PAM) may be added to improve treatment efficacy.

Compositions of the present invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as if further detailed above. The ability of PONl to sequester OP molecules, suggests use of same in the decontamination of OP contaminated surfaces and detoxification of airborne OP.

Thus, an aspect of the invention further provides for a method of detoxifying a surface contaminated with an OP molecule; or preventing contamination of the surface with OP. The method is effected by contacting the surface with PONl.

Thus, synthetic and biological surfaces contemplated according to embodiments of the invention include, but are not limited to, equipment, laboratory hardware, devices, fabrics (clothes), skin (as described above) and delicate membranes (e.g., biological). The mode of application will very much depend on the target surface. Thus, for example, the surface may be coated with foam especially when the surface comprises cracks, crevices, porous or uneven surfaces. Application of small quantities may be done with a spray-bottle equipped with an appropriate nozzle. If a large area is contaminated, an apparatus that dispenses a large quantity of foam may be utilized.

Coatings, linings, paints, adhesives sealants, waxes, sponges, wipes, fabrics which may comprise the PONl may be applied to the surface (e.g., in case of a skin surface for topical administration). Exemplary embodiments for such are provided in U.S. Pat. Application No. 20040109853.

Surface decontamination may be further assisted by contacting the surface with a caustic agent; a decontaminating foam, a combination of baking condition heat and carbon dioxide, or a combination thereof. Sensitive surfaces and equipments may require non corrosive decontaminants such as neutral aqueous solutions with active ingredient (e.g., paraoxonases).

In addition to the above described coating compositions, OP contamination may be prevented or detoxified using an article of manufacture which comprise the PONl immobilized to a solid support in the form of a sponge (as described above), a wipe, a fabric and a filter (for the decontamination of airborne particles). Chemistries for immobilization are provided in U.S. Pat. Application 20040005681, which is hereby incorporated in its entirety.

The terms "comprises", "comprising", "includes", "including", "having" and their conjugates mean "including but not limited to".

The term "consisting of means "including and limited to". The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases "ranging/ranges between" a first indicate number and a second indicate number and "ranging/ranges from" a first indicate number "to" a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Current Protocols in Immunology" Volumes I-III Coligan J. R, ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., Eds. (1984); "Animal Cell Culture" Freshney, R. I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PGR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.

EXAMPLE 1

Materials and Methods

Constructing PON1 gene libraries by random mutagenesis. Recombinant PON1 variant G3C9 (Gene bank entry: AY499193) was used as a template to create H115W and V346A amino acid mutations by primer designing. pET-Nes2-Bc and pET-Nesl-Fo primer (Table 9, below) was used to amplify 10 ng of template with a mutator Taq polymerase (mutazyme, Genemorph) in 25 μl of reaction mixture for 15 cycles. On average 1.7±0.65 amino acid mutations/gene with ~60% transition and -40% transversion were found. The PCR product was treated with Dpnl (to destroy the template plasmid), purified, and served as a template (10 ng) for another 15 cycles of nested PCR performed with Taq polymerase. The PCR products were digested with Ncol and Notl and cloned to pET32 vector with a C-terminal 6-His tag (Figure 1).

Constructing PON1 gene libraries by gene shuffling. The improved PON1 variants were separately amplified from their respective plasmids using Taq polymerase and primers pET-Nes2-Bc and pET-Nesl-Fo. To facilitate the removal of non- beneficial mutations, the PCR amplified wild-type PON1 gene was added at a 1:3 ratio to a mixture of PCR products from all the improved variants. Approximately 5 μg of purified DNA mixture in 50 μl reactions was digested with 0.01U DNasel (Takara) at 37°C for 2, 4, and 6 min. The reactions were terminated with 15μ1 of 0.5 M EDTA, and heating at 90 °C for 10 min, and were run on a 2% agarose gel. Fragments of 50-150 bps size were excised and purified using a gel extraction kit (Qiagen). The PON 1 gene was reassembled using 100 ng of purified DNA fragments and thermocycling in a 50 μl reaction mixture that contained 2.5 U Pfu Ultra (Stratagene). The cycling included: one denaturation step at 96 °C for 3 min, then 35 cycles composed of: (i) a denaturation step at 94 °C (30 s); (ii) nine successive hybridization steps separated by 3 °C each, from 65 °C to 41 °C, for 1.5 min each (total 13.5 min), and (iii) an elongation step of 1.5 min at 72 °C. Finally, a 10 min elongation step at 72 °C was performed. The assembly product was amplified by a nested PGR reaction with primers pET-Nesl-Bc and pET-NesO-Fo. In this step, 1 μl of the assembly reaction was used as a template in a standard 50 μl PGR reaction. The purified PGR product was digested with Ncol and Notl, and cloned into the pET32 vector with a C-terminal 6-His tag (Figure 1).

Constructing PON1 gene libraries by using designed oligonucletides at targeted positions. The PON 1 gene having H115W mutation was used as a template to construct a library using synthetic oligos by ISOR protocol. Briefly, H115W mutant gene was digested with DNasel. Approximately 5 μg of purified DNA in 50 μl reactions was digested with 0.01 U DNasel (Takara) at 37 °C for 2, 4, and 6 min. The reactions were terminated with 15 μl of 0.5 M EDTA, and heating at 90 °C for 10 min, and were run on a 2 % agarose gel. Fragments of 50-150 bps size were excised and purified using a gel extraction kit (Qiagen). The PON 1 gene was reassembled using 100 ng of purified DNA fragments with oligonucleotides encoded one mutation and 20 flanking nucleotides matching the PON1 gene (Table 9, below). Assembly PGR was performed in a 50μ1 reaction mixture that contained 2.5 U Pfu Ultra (Stratagene). The cycling included: one denaturation step at 96 °C for 3 min, then 35 cycles composed of: (i) a denaturation step at 94 °C (30 s); (ii) nine successive hybridization steps separated by 3 °C each, from 65 °C to 41 °C, for 1.5 min each (total 13.5 min), and (iii) an elongation step of 1.5 min at 72 °C. Finally, a 10 min elongation step at 72 °C was performed. The assembly product was amplified by a nested PCR reaction with primers pET-Nesl-Bc and pET-NesO-Fo. In this step, 1 μl of the assembly reaction was used as a template in a standard 50 μl PCR reaction. The purified PCR product was digested with Ncol and Notl, and cloned into the pET32 vector with a C-terminal 6-His tag (Figure 1). Double emulsion and sorting by FACS. Substitution libraries were sorted by compartmentalization of single E. coli cells, each expressing an individual library variant in double emulsion droplets, and sorting these droplets by fluorescent activated cell sorter (FACS), essentially as described (references are provided hereinbelow under the "Reference Section"). BL21 (DE3) cells possessing GFPuv gene in the genome were used for expression of the PON 1 under the T7 promoter. Plasmid DNA was transformed and grown while shaking at 250 RPM in 5ml 2xYT media containing 100μg/ml ampicillin and ImM CaCl₂, for 12 hrs at 30 °C, followed by another 24 hrs at 20 °C. The cells were centrifuged at 3000 g for 10 min at 4 °C, resuspended in 2xYT, and kept for lhr at room temperature. They were then rinsed twice in 0.1 M Tris-HCl, 1 mM CaCl₂, 0.1 M NaCl, pH 8.0, resuspended in the same buffer, and passed through a 5 μm filter (Sartorius). Filtered cells were compartmentalized in the first emulsion (water- in-oil), and 100 mM solutions of CMP-MeCyC racemate or DEPCyC was added to the oil phase (0.8 μl, to a final concentration of 50μΜ). The production of the second emulsion (water-in-oil-in-water) and sorting were performed as described. More than 10⁶ events, at 2000 events/sec, were stored using FACSAria (Becton-Dickinson) (Figure 2). Events corresponding to single E. coli cells were gated by GFP emission (at 530nm, using blue laser for excitation). Approximately 5000 events were sorted to 96- well plates containing 200 μl of 2xYT media (~1000 events per well). The plates were immediately moved to 37 °C, incubated for 1 hr while shaking at 250 rpm, plated on LB-agar plates containing 100μg/ml ampicillin and 20mM glucose, and grown overnight at 30 °C. Recovery of the sorted cells was determined by comparing the number of colonies on the LB plates to the number of events sorted by the FACS, and was found to be 20-40%.

Screens in 96-weIl plates. Randomly picked colonies were individually grown with shaking in 96-deep-wells plates, using 0.5 ml 2xYT medium containing 100 μg/ml ampicillin and 1 mM CaCl_¾ for 8 hrs at 30 °C, followed by another 16 hrs at 20 °C. Several repeats of wild-type PON1 were grown as controls. Following growth (OD_600nm =4), the plates were centrifuged at 3000 g for 15 mins at 4 °C, and pellets were kept at - 70 °C for few hours. The pellets were resuspended in 200 μl of lysis buffer (0.1M Tris- HCl pH 8.0, ImM CaCl₂, 10μg/ml lysozyme (Sigma), 0.2 % Triton x-100, and 5 units/ml benzonase (Novagen)), and lysed by shaking at 1300 rpm for 30 min at 37 °C. The pellet was removed by centrifugation at 4000 rpm for 20 min at 4 °C, and the supernatant was transferred to a new set of plates and stored at 4 °C.

Apparent enzymatic rates (vo) for different substrates were measured in a plate reader (Synergy-HT BioTek) using an appropriate volume of clarified lystaes (0.1-10 μl depending on the substrate). 25μΜ of IMP-MeCyC and CMP-MeCyC were used and release of coumarin was measured at 405 nm. In order to get S_p isomer, 10-20nM of 3B3 purified enzyme were used to cleave R_p isomer from the 25 μΜ of racemic mixture. All rates were determined at the linear range of product release, and background rates (lysates containing no PON1) were subtracted to give the observed initial rate (vo).

Enzymes purification and kinetics. Variants exhibiting the highest rates with the target substrate were grown in 50 ml cultures; cells were harvested by centrifugation, resuspended, and disrupted by sonication. Ammonium sulfate was added to the lysate to 5 5% (wt/vol). The precipitate was dissolved and dialyzed against activity buffer (Tris-HCl 50mM pH=8, CaCl₂ 1mM, NaCl 50mM, Tergitol 0.1%.) and purified on Ni-NTA (Novagen). Fractions were analyzed for paraoxonase activity and purity (by SDS-PAGE), pooled, dialyzed against activity buffer supplemented with 0.02 % sodium azide, and stored at 4 °C. Protein purity was typically 70-80 % by SDS- PAGE gel. Variants 4E9 and rePONl (G3C9) were further purified by FPLC purification using a mono-Q column (HiPrep 16/10 Q FF, GE healthcare) eluted by activity buffer with 250 mM NaCl, concentrated (vivaspin 20 MWCO 20KDa), loaded on a gel filtration column (HiLoad 26/60 Superdex 75, GE healthcare) and dialyzed against activity buffer supplemented with 0.02 % sodium azide for long term storage at 4 °C. Protein purity was assessed to be >97% by SDS-PAGE gel. A range of enzyme concentrations (0.01-4 μΜ) and substrate concentrations was applied (from 0.3 x KM up to 2-3 x Km). Product formation was monitored spectrophotometerically in 96-well plates with 200-μ1 reaction volumes. For each purified variants, at least three independent repeats were done for kinetic parameters and values were determined by fitting the data directly to the Michaelis-Menten using KaleidaGraph (Figure 3). The catalytic activity of evolved variants with R_p-CMP-coumarin was obtained by measuring kinetics of the 2^nd phase observed with racemic CMP-coumarin after consumption of the S_p-CMP-coumarin by one of the S_p evolved variants (e.g. variant OC9 at 30nM). By measuring initial rates using several substrate concentrations, we could estimate the apparent k_cat/K_M for this isomer.

Conversion of CMP-coumarin to GF. Caution: Although the total amount of the in situ generated cyclosarin (GF) in aqueous solution is non-hazardous, the reader should be aware of its high potency as inhibitor ofAChE. CMP-coumarin (0.5 ml of 1 mM) was incubated at room temperature in 0.2 M NaF pH 5.0. The quantitative and completion of the conversion of CMP-coumarin to GF (about 30 min) was verified by monitoring the release of the coumarin leaving group, using 100-fold dilution in 50 mM Tris-buffer pH 8 and measuring the absorption at 400 nm (the molar absorption of free coumarin was 3.7xl0⁴ M_-1cm^-1). The observed release of the coumarin was >95% of the calculated value. ³¹P-NMR also indicated the complete and full conversion of CMP- coumarin into CMP-F. This was apparent from the disappearance of the ³¹P signal attributed to CMP-coumarin (δ, 29.8 ppm) and the concomitant rise of a doublet centered at δ, 32.2 ppm with a J_p-F value of 1045 Hz which is typical to O-alkyl methylphsphonofluoridates. To determine the concentration of the GF stock solution produced in 0.2 M NaF, a ~10⁵-fold dilution into a known concentration (~ 5 nM) of Torpedo californica AChE (TcAChE) in 50 mM phosphate buffer pH 8.0, permitted to titrate the content of the toxic S_p isomer of GF, which was found to be 0.43-0.48 mM (ca. half of the racemic GF generated from ImM racemic CMP-Coumarin). The conversion of the IMP-, and PNP-Coumarin analogs to the respective fluoridates, and the determination of the kinetic parameters, was similarly performed and monitored.

The formation of GF, and the irreversibility of the conversion process, were further validated by three experiments. Firstly, the bimolecular rate constant of the toxic isomer of the CMP-coumrin analog when inhibiting TcAChE was 3.3xl0⁷ M^" min . In contrast, the insitu generated GF exhibited a bimolecular rate constant of 1.3xl0⁹ M^" ¹min^-1). The 40-fold increase in the inhibition potency of TcAChE is consistent with what would be expected from the inhibition rate constant of AChE by authentic GF. Secondly, the half-life of the in situ generated GF in activity buffer (50 mM Tris, 1 mM CaCl₂, 0.1 % Tergitol, pH 8.0) is 85 to 130 min (based on loss of anti-AChE activity; see below), while t1_/2 of Sp-CMP-coumarin under the same conditions is ~ 530 min. (The in situ generated GF solution were therefore freshly made before screening and kept in an ice bath until used). Thirdly, since the released coumarin was present in the reaction mixture, we aimed to exclude the possibility that the conversion is reversible, and that we were actually monitoring PONl-mediated hydrolysis of CMP-Coumarin and a subsequent shift in equilibrium. Therefore the effect of an excess of free coumarin on the hydrolysis of in-situ generated GF was tested (Figure 4). The above described assay was repeated with 4 additional equivalents of coumarin (one equivalent was generated by the fluoride exchange). The results indicated a small reduction in catalytic activity (1.15xl0⁷ to 0.96xl0⁷ M^-1 min ^-1) of 4E9 on GF. Because reversibility is expected to result in higher rates in the presence of excess of coumarin, this result indicates that the reverse conversion of GF into CMP-coumarin does not occur under the assay conditions.

Determination of kcat/ΚΜ values with in-situ generated GF. The GF stock was diluted 1000-fold in cold distilled water and then further diluted 20-fold into the 0.005 to 0.2 μΜ PON variant in activity buffer (Tris-HCl 50mM pH=8, CaCl₂ ImM, NaCl 50mM, Tergitol 0.1%.). The nominal racemic GF concentration was set to 40-50 nM. At various time intervals, the reaction mixture is diluted 10- fold into 2.5 nM TcAChE in 50 mM phosphate buffer pH 8,0, 25°C. The phosphate buffer that chelates calcium, and the dilution, quenched the PON1 activity with GF. Residual TcAChE activity was measured after 10 and after 20 min (to ascertain completion of inhibition) by aliquating 10 μl into 1 ml Ellman assay solution containing 1 mM acetylthiocholine as substrate. The %-inhibition of TcAChE by the same GF solution with out PON was considered as 100% anti-AChE potency attributed to the toxic isomer of GF. This %- inhibition decreased over time of incubation with PON1, and k_obs was calculated by fitting the %-loss of anti-TcAChE potency versus time to a mono-exponential equation. The concentration of PON1 was set so that degradation of >50% of GF (i.e., gain of 50% AChE activity) occurred within less than 10 mins (although this was impossible with the poorly active variants such as wild-type-like rePONl-G3C9).

AChE protection assays. The assays were performed by pre-incubation of the PON1 variant and the OP (as exemplified in the below protocol for CMP-Coumarin), or by direct competition of the PON1 variant and AChE, as exemplified in the second protocol with CMP-F. Briefly, randomly picked colonies of library variants were grown and lysed as above. Clarified cell lysates were diluted 1:4 in activity buffer, and 50μ1 diluted lysate were mixed with ΙΟμΙ of 6μΜ CMP-coumarin. The reactions were incubated (15 mins), and an equal volume of AChE solution (0.25 nM AChE, in PBS, 0.1% BSA) was added. Following 15 min's incubation, samples (20μ1) were mixed with Ellman's reagent and the AChE substrate (180μ1, 0.85mM DTNB, 0.55mM acetylthiocholine, in PBS), and initial rates were measured at 412nm. Residual AChE activity was determined by comparing initial rates those without OP. The screen with CMP-F was performed with the following modifications: undiluted cell lysates were mixed with an equal volume of AChE solution (0.5nM), and freshly made CMP-F was added to 1μΜ final concentration. Reactions were incubated for 15 mins, and residual AChE activity was determined as above. The catalytic specificity k_cat/K_M of purified variants was measured by mixing the in situ prepared OP-fluoridates (40nM) with purified PONl variants (0.1-0.01μΜ) in activity buffer. Samples of this reaction mix were taken at different times, diluted (1:10) with the AChE solution (4nM AChE, 0.1% BSA, ImM EDTA, in PBS), incubated for 15 mins, and residual AChE activity was determined as above. The apparent k_cat/KM values were derived from the slope of the resulting single exponential curve.

Prophylactic activity of 4E9 in a mouse model. Eight weeks old male mice of strain C57BL/6J strain, were supplied under germ-free conditions by the Animal Breeding Center of The Weizmann Institute of Science (Rehovot, Israel). The mice were housed in a light- and temperature-controlled room. All animals were handled according to the regulations formulated by the Institutional Animal Care and Use Committee (application number 04590909-2). Prior to treatments, blood samples were taken (50-75μ1, retero-orbital) into heparin (ΙΟμΙ ,1:10). Mice were then weighted (average weight 24.5(gr) ± 2.2) and PONl variant 4E9 or rePONl (210-260 μg/ml, >97% pure in isotonic activity buffer: Tris 50mM pH=8, CaC12 ImM, NaCl 100mM, tergitol 0.02%) were injected i.v to the tail vein at different doses(l.l, 2.1 or 2.2 mg/kg). After 55' or 5h55' blood samples were obtained as described and mice were reweighed. After 1 or 6 hours, intoxication was induced by a single i.v. administration of Sp-CMP- coumarin (26.5 ^g/ml], PBS) at a dose of 290 μg/Kg. All animals were observed closely for clinical signs following CMP-coumarin intoxication during the first 24 hours and were kept for at least 14 days before sacrifice. Control mice were injected i.v. to the tail vein with either: isotonic activity buffer (200 μl) or Atropine sulfate [20 mg/kg] and 2-PAM [25 mg/kg] in PBS just 5 min prior to intoxication as indicated. The toxicity of PON1 variant 4E9 or the isotonic activity buffer were assayed by injecting them to mice without an OP challenge, as described, and monitoring for at least 14 days. All clinical signs noted following Sp-CMP-coumarin intoxication were categorized to mild, moderate or severe reactions. Mild reactions were characterized by straub tail and ataxia. Moderate reactions consisted in addition decreased motor activity and tremors while animals with severe reactions exhibited in addition ventral position, fasciculation and dyspnea as well. The overall reactions observed following Sp-CMP-coumarin intoxication were scored using semi-quantitative grading of five grades (0-4), taking into consideration the severity of the reactions (0 = No Reactions, 1 = Mild Reactions,2 = Moderate Reactions, 3 = Severe Reactions, 4 = Mortality).

EXAMPLE 2

Directed evolution of PON1 for S_P-CMP hydrolysis

Several variants of rePONl with an enhanced activity towards a racemic mixture of CMP-Coumarin were previously isolated by screening 'neutral drift' libraries of rePONl (e.g. 1G3, 2G9). The most active variant was found to be 3B3 with ~250-fold higher catalytic efficiency ( k_cat/K_M 20xl0⁶ M^-1min^-1) compared to the wild-type-like rePONl (k_cat/K_M 0.08xl0⁶ M^-1min^-1; Table lb; Table 2 below). Although the hydrolysis by rePONl-3B3 was also restricted to the R_p isomer (Figure 6a), the high catalytic efficiency and ^-stereoselectivity coulf be used to isolate the Sp isomers of CMP- coumarin and IMP-coumarin from the corresponding racemates, and apply them for the subsequent screens. Low activity of rePONl mutants H115W and V346A rePONl towards Sp-CMP-coumarin was also identified. However, their activity with Sp-CMP- coumarin was too low for detection under library screening conditions. Therefore the first rounds IMP-coumarin, a less bulky G-agent analogue whose Sp isomer is more reactive with PON1 were used (Table 2, below).

Random mutagenesis of rePONl-H115W-V346A and screening of the resulting library in 96-well plates with Sp-IMP-Coumarin yielded several improved variants that typically carried one mutation in addition to H115W and V346A (Table 3, below). A second round of mutagenesis and screening with Sp-IMP-Coumarin led to the isolation of variants in which V346A was removed and the H115W and F222S mutations dominated (Table 3, below). As the evolving variants became more reactive with the Sp isomer, the 3^rd generation library could be screened with both S_p-IMP- and Sp-CMP-Coumarin. Indeed, this round resulted in several variants with improved activities towards S_P-CMP-Coumarm (e.g. 3A7, 8C8; Table lb; Table 4, below). However, since the 4^th round of mutagenesis and screening yielded no further improvements, a structure-based targeted library was designed and subjected it to high- throughput screening (>10 variants per run) by FACS sorting, as described below.

EXAMPLE 3

Highly proficient variants by FACS screening of double emulsion droplets.

The targeted substitutions library was based on PONl's active-site structure. In particular, a recently obtained crystal structure of the re-G3C9-H115W indicated movements of several side-chains in response to this mutation, including those of residues 69, 134 which are in direct contact with W115, and of the more remote residues 346, 347 and 348. therefore a library was generated by randomizing these positions and those of residues 115 and 222 that were found to be mutated in all active variants of the 3^rd round (Table 4, below). An oligo spiking strategy was used that incorporated the randomizing oligos onto re-PONl-H115W in a combinatorial manner so that each library variant carried on average 4 mutated positions. Due to the intense level of mutagenesis, and the targeting of the active-site, the libraries comprised mostly inactive variants. To purge inactive library clones, we employed a high-throughput FACS screen using a fluorogenic phosphotriester dubbed DEPCyC that was found to correlate well with the activity with Sp-CMP-Coumarin¹². E. coli cells transformed with the plasmid library were compartmentalized in water-in-oil emulsion droplets. The fluorogenic substrate was added, and the primary emulsion droplets were converted to double-emulsion droplets that were sorted by FACS. Cells in isolated droplets were plated, picked and assayed in 96-well plates for Sp-CMP-coumarin activity.

The most active variants isolated from this 5^th round (Table 5, below) carried mutations L69G/A and H134R, in addition to H115W and F222S that appeared in the previous rounds. These variants were shuffled, together with random mutagenesis at low rates (~1.7 amino acid exchanges per gene), and the resulting 6^th round library was sorted by FACS, and then screened in 96-well plates. The most improved variants carried five key mutations: L69G, H115W, H134R, F222S and T332S (Table 6, below). The best variant, 3D8 exhibited a k_catIK_M value of 1.2xl0⁷ M^-1rnin^-1 with S_p-CMP- coumarin (Table 1 below; Figure 6a). Further, 3D8 and other variants from the 6^th round exhibited similar rates with both the S_p and R_p isomers as indicated by the complete hydrolysis of the racemic CMP-coumarin with monophasic kinetics (Figure 6a). Figure 6b shows how the toxic isomer (S_p) of a Cyclosarin coumarin analogue called CMP is hydrolyzed by different variants and the wild-type like rePONl (G3C9) with time. We added the purified variants and the substrate at the indicated amounts and followed the hydrolysis by reading the increase in absorption of the coumarin leaving group with time. As can be seen, both rePONl and 3B3 can hardly hydrolyze the substrate while 4E9 and 3D8 do so readily. Figure 6c show how these variants hydrolyze the in-situ generated agent (Cyclosarm) termed here CMP-F as it is the fluoride derivative of CMP. Purified variants and the substrate were added at the indicated amounts and followed the hydrolysis by sampling the reaction at different time points and adding the samples to purified AChE. The residual AChE activity was used as a measure of the amount of substrate hydrolyzed sine they are correlated. As can be seen, variant 3B3 is the worst while 4E9 is the best hydrolyzer. By fitting the curve to a first-order equation we can derive the apparent rate constant for the hydrolysis of GF by these variants.

EXAMPLE 4

Acetylcholinesterase protection assay

Along the selection for variants with higher rates, the concentration of the CMP- coumarin substrate was decreased to enable the isolation of variants with improved K_M as well as k_cat. However, to be able to screen at concentrations that correspond to the very low toxic concentrations of cyclosarin in vivo (~1 μΜ)^{3, 20} , and for variants that efficiently degrade cyclosarin itself a screen was developed based on monitoring the rescue of AChE, the OP's physiological target. AChE was added to crude bacterial lysates expressing the library PON1 variants. The OP was added, and the residual activity of AChE was subsequently measured using a chromogenic assay to indicate the level of OP degradation by the tested variant. The FACS sorted 6^th round library was re- screened in 96-well plates using the AChE assay and 1 μΜ of racemic CMP-coumarin. 730 randomly-picked colonies were screened and 13 variants were isolated with improved activity by 2-12 fold relative to 3D8 (Table 7, below). Although at this stage variants that exhibited sufficiently high catalytic proficiency were identified, these were selected and tested with coumarin surrogates. In fact, the fluoride leaving group of the actual threat agents substantially differs from coumarin (Figure 5) - fluoride is more reactive and is a much smaller leaving group. However, the toxicity of nerve agents prevents their use in ordinary labs. Therefore a non- hazardous screening protocol was developed based on CMP-F generated in situ, in dilute aqueous solutions, by replacing the coumarin leaving group of CMP-coumarin with fluoride. This exchange was spectroscopically monitored by following the release of the coumarin. The inhibition of TcAChE by in situ generated CMP-F proceeded 40- fold faster than with S_p-CMP-Coumarin, with the expected of 1.3xl0⁹ M^-1min ^-1. Using the in situ generated CMP-F, we measured the k_cat/KM values of the evolved variants under pseudo-first-order conditions (CMP-F <50 nM, well under a likely KM)- Encouragingly, the activities with the coumarin and fluoridate Sp isomers were comparable, and at least three variants (0C9, 2D8, 1A4) exhibited k_cat/KM values of >10⁷ M^min^"1 with CMP-F (Table 8, below). The AChE protection assays therefore confirmed the ability of the evolved variants to protect AChE from cyclosarin in vitro, and validated the coumarin analogues as faithful surrogates of the actual G-agents. The assay also confirmed that the starting point, rePONl-G3C9 is much more active with CMP-F than CMP-Cuomarin (Table lb), as is human PON1, although the k_cat/K_M (~10⁵ M^-1min^-1) is >100-fold too low for in vivo detoxification using reasonable amounts of enzyme.

The evolved variants were sufficiently active to enable library screens using the in situ generated agent. This approach is highly attractive, since the assay of AChE protection against the actual threat agent mimics the in vivo protection challenge whereby the catalytic scavenger must be sufficiently active to intercept the threat agent before the latter reacts with AChE. Therefore, the 13 most improved variants from the last round were re-screened using CMP-F at the expected plasma concentration for lxLD₅o exposure (1 μΜ). Nine variants exhibited improved activities relative to 3D8 (Table 7, below). Of these, 3 variants (4E9, 5F3 and 6A3) exhibited the highest specific activity upon examination of the amount of soluble expressed protein. Following sequencing and protein purification, 4E9 was identified as the most active variant with both Sp-CMP-coumarin and S_P-CMP-F (k_cat/K_M = 2.23xl0⁷ M^-1rnin^-1, and 1.7xl0⁷ M^-1min^-1,_. respectively; Table lb below).

EXAMPLE 5

Prophylactic protection assays

To validate that hydrolysis of the toxic isomer by a variant with k_cat/K_M values of >10⁷ M- min^" should protect against lethal OP exposure at a low protein dose, rePONl- 4E9 was tested as a prophylactic in a mouse model. Due to safety issues, the CMP- Cuomarin surrogate was applied, but the challenge was upgraded by using the toxic somer only (S p-CMP-coumarin) and by administrating it directly by i.v. injection. The results indicated a survival rate of 45% for mice pretreated with 1.1 mg/kg 4E9 one hour prior to the OP exposure (Table 10 below). Increasing the 4E9 dose to 2.2 mg/kg increased the percent of surviving animals to 75%, supporting the predicted correlation between k_cat/K_M and in vivo and protection level . Twenty-four hours after exposure, the survival rate was 75% for mice receiving 4E9 either one, or six hours before the OP challenge. A similar survival ratio (63-75%) was observed 14 days later. As expected, the wild-type-like rePONl-G3C9 (estimated k_cat/K_M for S_P-CMP-coumarin <2xl0² M- ¹min^"1), which served as a starting point for the directed evolution of 4E9, conferred no protection. Notably, treatment of mice with atropine and 2-PAM, even 5 minutes prior to challenge, gave very poor protection against the cyclosarin coumarin surrogate, as is the case with cyclosarin itself: the 24h survival was only 22%, and there was no survival 96h post challenge. Further, whereas 4E9 protected mice exhibited only mild intoxication symptoms 2-12h after the challenge, all atropine plus 2-PAM treated mice displayed severe intoxication symptoms with no improvement until death.

a. Annotation of variants: The first digit relates to the plate number, and the following letter-digit to its location within this plate. For example, variant 3A7 = plate #3, well A7; n.d., not detectable.

b. Denoted in bold are mutations in active-site residues.

c. Enzymatic parameters were measured with purified proteins and comprise the average obtained from the 3 independent repeats. Error ranges represent the standard deviations observed between measurements. A more complete set of parameters including separate k_cat and K_M values when available, are provided in the Tables below. Values in parentheses describe the fold-change compare to the starting point, rePON-G3C9. The kinetic parameters for Sp-CMP-coumarin were spectrophotometerically measured with pure substrate samples¹⁵. Parameters for R_p- CMP-coumarin were determined with the racemate, and for CMP-F with the in situ prepared substrate and an AChE inhibition assay (see Methods for details).

d. The catalytic efficiency was estimated in the reference section

e. Variants exhibited a single-phase kinetics of product release when reacted with racemic CMP-coumarin, suggesting that the rates of hydrolysis for R_P-and S/>-CMP-coumarin are similar.

a. The annotation of the variants: The first letter relates to the plate number, and the letter- digit to the location of the clone within this plate. For example, variant 1G3= plate # 1, well G3.

b. Round of mutagenesis and screening

c. Shown are all variants that exhibited higher S_p-IMP-coumarin activity in crude cell lysates relative to the H115 W + V346A PONl mutant. For each variant, enzymatic activities were measured in crude lysate and denoted are the average values of fold improvement obtained from 3 independent repeats. The values had s.d.<20% of their value.

Non-synonymous mutations observed in each variant. Mutations in active site residues are noted by underlined.

e. These two variants had the same amino acid exchanges. The small differences in activity may relate to differences in the composition and number of synonymous mutations.

The annotation of the variants: The first letter relates to the plate number, and the letter-digit to the location of the clone within this plate. For example, variant 4D2= plate # 4, well D2.

Shown are all variants that exhibited higher S_p -IMP-coumarin and S_p -CMP-coumarin activities in crude lysates relative to the H115W + V346A PON1 mutant. For each variant, enzymatic activities were measured in crude lysate and denoted are the average values of fold improvement obtained from 3 independent repeats. The values had s.d.<20% of their value.

Non-synonymous mutations observed in each variant. Mutations in active site residues are noted by underline.

a. The annotation of the variants: The first letter relates to the plate number, and the letter-digit to the location of the clone within this plate. For example, variant 2F8= plate # 2, well F8. .

b. Shown are all variants that exhibited higher S_p-CMP-coumarin activities in crude lysates relative to the 8C8 mutant. For each variant, enzymatic activities were measured in crude lysate and denoted are the average values of fold improvement obtained from 3 independent repeats. The values had s.d.<20% of their value.

c. Non-synonymous mutations observed in each variant. Mutations in active site residues 0 are underlined.

Table 8: Activities of selected rePONl variants on Sp-CMP-coumarin and its a,b,c,d

a. The figures shown are values of ko_at/K_Mxl0 (M^-1 min^-1 )

b. Data for OP-coumarin are based on release of the chromophore monitored at 400 nm. c. The k_cat/Km values for the fluoridates were determined by monitoring the rate of loss of anti-AChE potency of the in situ-generated compound, assuming K_m»[P-F]. Calculations are based on a single enzyme concentration selected to fit the dynamic range for determination of the apparent k_obs of loss of anti-AChE potency.

The coumarin leaving group was replaced by fluoride in racemic CMP -coumarin to yield the racemic fluoridates of CMP (CMP-F). Note that the data for the hydrolysis of CMP-F can be attributed mostly to the toxic (Sp) isomer of CMP-F.

k_cat/K_m for Round 3 best mutants . G agents were at 0.5 μΜ and the enzymes at concentrations well below the OP, thus fulfilling the catalytic conditions for the hydrolysis of the nerve agents. Data shown (μΜ ^-1min ^-1) , mean ±SD, n=3.

1. A significant systematic improve across all G agents, when compared to libraries Gl and G2

2. The best variant so far is 1-I-Fll.

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.

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Claims

WHAT IS CLAIMED IS:

1. An isolated polypeptide comprising an amino acid sequence of serum paraoxonase (PONl) .having catalytic efficiency of k_catIK_M ~10⁶ M min^-1 for a nerve- agent substrate.

2. The isolated polypeptide of claim 1, wherein said nerve-agent substrate comprises an Sp isomer.

3. The isolated polypeptide of claim 1, having catalytic efficiency of k_cat/K_M ~10⁷ M^{- 1} min^-1 for Sp nerve-agent substrates.

4. The isolated polypeptide of claim 1, wherein said amino acid sequence of serum paraoxonase (PONl) comprises a mutation selected from the group consisting of: L69G/A/L/V/S/M, K70A/S/Q/N, Y71/F/C/A/L/I, H115W/L/V/C, H134R/N, F222S/M/C, F292S/V/L, T332S/M/C/A, M196V/L/F, V97A ,V346A ,N41D, Y293S, V97A, V276A , T326S, SUIT, SHOP, P135A, N41D, N324D, M289I, L240S/V, L14M, L10S, K233E, H285R, H243R,, F28Y, F264L, D309N/G, A6E, N227L, F178V,D49N, wherein amino acid coordinates correspond to the G3C9 PONl variant.

5. The isolated polypeptide of claim 1 being expressible in bacteria.

6. The isolated polypeptide of claim 1, wherein said amino acid sequence is selected from the group consisting of the sequences set forth in SEQ ID NO: 129, 2-54 and 120-128.

7. The isolated polypeptide of claim 1, wherein said amino acid sequence is selected from the group consisting of the sequences set forth in SEQ ID NO: 129, 2, 4, 7, 9, 12, 24, 47, 53, 120-128.

8. An isolated polynucleotide comprising a nucleic acid sequence encoding the polypeptide of any of claims 1-6.

9. A pharmaceutical composition comprising as an active ingredient the isolated polypeptide of any of claims 1-6 and a pharmaceutically acceptable carrier.

10. A nucleic acid construct comprising the isolated polynucleotide of claim 8 and a cis-regulatory element driving expression of said polynucleotide.

11. A method of treating or preventing organophosphate exposure associated damage in a subject in need thereof, the method comprising providing the subject with a therapeutically effective amount of the isolated polypeptide of any of claims 1-8 to thereby treat the organophosphate exposure associated damage in the subject.

12. The method of claim 11, wherein said providing is effected prior to the organophosphate exposure.

13. The method of claim 11, wherein said providing is effected by inhalation administration.

14. The method of claim 11, wherein said providing is effected 10 hours prior to the exposure until 7 days following exposure.

15. The method of claim 11, wherein said providing is effected by inhalation and injection.

16. The method of claim 11, further comprising administering to the subject atropine and optionally oxime.

17. The method of claim 11, wherein said providing is effected by topical application.

18. An article of manufacture for treating or preventing organophosphate exposure associated damage, the article of manufacture comprising the isolated polypeptide of any of claims 1-8 immobilized on to a solid support.

19. The article of manufacture of claim 18, wherein said solid support is for topical administration.

20. The article of manufacture of claim 19, wherein said solid support for topical administration is selected from the group consisting of a sponge, a wipe and a fabric.

21. The article of manufacture of claim 18, wherein said solid support is selected from the group consisting of a filter, a fabric and a lining.

22. A method of detoxifying a surface, the method comprising contacting the surface with the isolated polypeptide of any of claims 1-8, thereby detoxifying the surface.

23. The method of claim 22, further comprising contacting the surface with a decontaminating foam, a combination of baking condition heat and carbon dioxide, or a combination thereof.

24. The method of claim 22, wherein said polypeptide is comprised in a coating, a paint, a non-film forming coating, an elastomer, an adhesive, an sealant, a material applied to a textile, or a wax.