EP2478093A2 - Polypeptides pon1 isolés, polynucléotides codant pour ceux-ci et utilisations de ceux-ci dans le traitement ou la prévention des dommages associés à l'exposition à un organophosphate - Google Patents

Polypeptides pon1 isolés, polynucléotides codant pour ceux-ci et utilisations de ceux-ci dans le traitement ou la prévention des dommages associés à l'exposition à un organophosphate

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Publication number
EP2478093A2
EP2478093A2 EP10768078A EP10768078A EP2478093A2 EP 2478093 A2 EP2478093 A2 EP 2478093A2 EP 10768078 A EP10768078 A EP 10768078A EP 10768078 A EP10768078 A EP 10768078A EP 2478093 A2 EP2478093 A2 EP 2478093A2
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EP
European Patent Office
Prior art keywords
isolated polypeptide
cmp
coumarin
variants
pon1
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP10768078A
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German (de)
English (en)
Inventor
Dan S. Tawfik
Rinkoo Devi Gupta
Moshe Goldsmith
Yaacov Ashani
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Yeda Research and Development Co Ltd
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Yeda Research and Development Co Ltd
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Publication of EP2478093A2 publication Critical patent/EP2478093A2/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)

Definitions

  • 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 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.
  • 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.
  • an isolated polypeptide comprising an amino acid sequence of serum paraoxonase (PON1) having catalytic efficiency of k cat IKM ⁇ 10 6 M -1 min -1 for a nerve- agent substrate.
  • PON1 serum paraoxonase
  • the nerve-agent substrate comprises an Sp isomer.
  • the isolated polypeptide has catalytic efficiency of k cat IKM ⁇ 10 7 M ⁇ min -1 for Sp nerve-agent substrates.
  • the amino acid sequence of serum paraoxonase 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
  • the isolated polypeptide is expressible in bacteria.
  • 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.
  • 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.
  • an isolated polynucleotide comprising a nucleic acid sequence encoding the polypeptide.
  • a pharmaceutical composition comprising as an active ingredient the isolated polypeptide and a pharmaceutically acceptable carrier.
  • nucleic acid construct comprising the isolated polynucleotide and a cis- regulatory element driving expression of the polynucleotide.
  • a method of treating or preventing organophosphate exposure associated damage in a subject in need thereof comprising providing the subject with a therapeutically effective amount of the isolated polypeptide to thereby treat the organophosphate exposure associated damage in the subject.
  • the providing is effected prior to the organophosphate exposure.
  • the providing is effected by inhalation administration.
  • the providing is effected 10 hours prior to the exposure until 7 days following exposure.
  • the providing is effected by inhalation and injection.
  • the method further comprises administering to the subject atropine and optionally oxime.
  • the providing is effected by topical application.
  • an article of manufacture for treating or preventing organophosphate exposure associated damage comprising the isolated polypeptide immobilized on to a solid support.
  • the solid support is for topical administration.
  • the solid support for topical administration is selected from the group consisting of a sponge, a wipe and a fabric.
  • the solid support is selected from the group consisting of a filter, a fabric and a lining.
  • a method of detoxifying a surface comprising contacting the surface with the isolated polypeptide, thereby detoxifying the surface.
  • the method further comprises contacting the surface with a decontaminating foam, a combination of baking condition heat and carbon dioxide, or a combination thereof.
  • 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.
  • a coating a paint, a non-film forming coating, an elastomer, an adhesive, an sealant, a material applied to a textile, or a wax.
  • 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 ⁇ l , to a final concentration of 50 ⁇ ).
  • the production of the second emulsion (water-in-oil-in-water) and sorting were performed as described 4 . More than 10 6 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 ⁇ for 8C8, and 12.5 nM for 0C9 and 3D8. Substrate concentrations were varied from 0.4 ⁇ up to 1000 ⁇ .;
  • 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).
  • GF cyclosarin
  • GB sarin
  • GD soman
  • GD pinacolyl
  • 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 (12 ⁇ ) in the presence of variants 4E9, 3D8, 3B3 (plus addition of 0.03 ⁇ 4E9 after 6 mins; indicated by the black arrow), and wild-type-like rePONl (plus addition of 0.03 ⁇ 4E9 after 20 mins).
  • Figure 6B Hydrolysis of Sp-CMP-coumarin (6 ⁇ ) in the presence of variants 4E9, 3D8, 3B3, and rePONl.
  • Figure 6C Hydrolysis of Sp-CMP-coumarin (6 ⁇ ) in the presence of variants 4E9, 3D8, 3B3, and rePONl.
  • 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.
  • Organophosphates including pesticides and nerve agents, comprise a prime target for detoxification.
  • OPs Organophosphates
  • no natural enzymes are available that proficiently degrade most of these xenobiotics.
  • 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 ⁇ 10 7 M -1 min -1 ).
  • 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.
  • 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 ⁇ 3xl0 5 M ⁇ min 1 , and for soman (Pin-F), 7.4xl0 6 M -1 min -1 , and 0.58xl0 6 M ⁇ min -1 , for the two toxic isomers respectively).
  • an isolated polypeptide comprising an amino acid sequence of serum paraoxonase (PONl) having catalytic efficiency of k cat /KM ⁇ 10 6 M -1 min -1 for a nerve-agent substrate.
  • PONl serum paraoxonase
  • PONl serum paraoxonase
  • HDL high-density lipoprotein
  • OP organophosphorus
  • PONl has also been shown to be involved in the metabolism of lactones and cyclic carbonates.
  • 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.
  • additional polymorphisms in the coding region, 5' regulatory region, and PONl introns have been reported.
  • PONl Any PONl may be used e.g., human PONl, rabbit PONl. Others are listed below (Table lal).
  • 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).
  • a “nerve agent” refers to an organophosphate (OP) compound such as having an acetylcholinesterase inhibitory activity.
  • OP organophosphate
  • 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).
  • 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.
  • CWA chemical warfare agents
  • a CWA may be in airborne form and such a formulation is known herein as an "OP-nerve gas.”
  • 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.
  • 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
  • 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,
  • in vitro evolution process refers to the manipulation of genes and selection or screening of a desired activity.
  • 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.
  • 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, D
  • 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.
  • PON1 serum paraoxonase
  • 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).
  • 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).
  • 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).
  • 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.
  • the isolated polypeptide is selected from the list below (Table la2). Other polypeptides are listed in the Examples section which follows.
  • isolated refers to isolated from the natural environment e.g., serum.
  • polypeptide 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.
  • Peptide bonds (-CO-NH-) within the peptide may be substituted, for example, by
  • Synthetic amino acid substitutions may be employed to improve stability and bioavailability.
  • the present teachings also provide for nucleic acid sequences encoding such PON1 polypeptides.
  • an isolated polynucleotide including a nucleic acid sequence, which encodes the isolated polypeptide of the present invention.
  • 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).
  • 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.
  • genomic polynucleotide sequence refers to a sequence derived (isolated) from a chromosome and thus it represents a contiguous portion of a chromosome.
  • 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.
  • 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. 3:1671-1680 and Brogli et al., (1984) Science 224:838-843, Gurley et al. (1986) Mol. Cell. Biol.
  • 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.
  • a cis-regulatory sequence suitable for directing constitutive or inducible transcription in the host cells, as further described hereinbelow.
  • 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.
  • sequences i.e., tags
  • 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.
  • 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].
  • prokaryotic or eukaryotic cells can be used as host-expression systems to express the polypeptide coding sequence.
  • host-expression systems 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.
  • 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.
  • 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.
  • recovery of 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.
  • a method of treating or preventing organophosphate exposure associated damage in a subject in need thereof 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.
  • 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.
  • 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.
  • organophosphate compound refers to a compound comprising a phosphoryl center, and further comprises two or three ester linkages.
  • the type of phosphoester bond and/or additional covalent bond at the phosphoryl center classifies an organophosphorus compound.
  • the OP compound is known as an "oxon OP compound” or "oxon organophosphorus compound.”
  • the OP compound is known as a "thion OP compound” or "thion organophosphorus compound.”
  • 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.
  • an OP compound comprises an organophosphorus nerve agent or an organophosphorus pesticide.
  • 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.
  • the method is effected by providing the subject with a therapeutically effective amount of the PONl polypeptide of the invention.
  • PONl 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.
  • GI gastro-intestinal
  • 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.
  • the porous support may be formed into a typical household sponge, wipe or a towelette.
  • 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.
  • PONl may be administered to the subject per se or in a pharmaceutical composition where it is mixed with suitable carriers or excipients.
  • 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.
  • active ingredient refers to the PON1 accountable for the biological effect.
  • pharmaceutically acceptable carrier refers 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.
  • excipient refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient.
  • excipients examples include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
  • 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.
  • Topical administration is also contemplated according to the present teachings.
  • 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.
  • 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.
  • 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.
  • physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • 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).
  • 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.
  • 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.
  • 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.
  • the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.
  • compositions may take the form of tablets or lozenges formulated in conventional manner.
  • 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.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide.
  • 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.
  • compositions 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.
  • 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.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.
  • a suitable vehicle e.g., sterile, pyrogen-free water based solution
  • 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.
  • 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.
  • 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.
  • the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays.
  • 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).
  • MEC minimum effective concentration
  • 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.
  • 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.
  • compositions 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.
  • 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.
  • PONl to sequester OP molecules
  • 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.
  • 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.
  • 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).
  • 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).
  • 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.
  • compositions, methods 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.
  • a compound or “at least one compound” may include a plurality of compounds, including mixtures thereof.
  • 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.
  • 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.
  • 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).
  • PON1 gene libraries by gene shuffling 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.
  • 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 ⁇ g of purified DNA mixture in 50 ⁇ l reactions was digested with 0.01U DNasel (Takara) at 37°C for 2, 4, and 6 min.
  • the reactions were terminated with 15 ⁇ 1 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 ⁇ l 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.
  • H115W mutant gene was digested with DNasel. Approximately 5 ⁇ g of purified DNA in 50 ⁇ l reactions was digested with 0.01 U DNasel (Takara) at 37 °C for 2, 4, and 6 min. The reactions were terminated with 15 ⁇ l 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 50 ⁇ 1 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.
  • the assembly product was amplified by a nested PCR reaction with primers pET-Nesl-Bc and pET-NesO-Fo. In this step, 1 ⁇ l of the assembly reaction was used as a template in a standard 50 ⁇ l 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”).
  • FACS fluorescent activated cell sorter
  • 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 100 ⁇ g/ml ampicillin and ImM CaCl 2 , 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 CaCl 2 , 0.1 M NaCl, pH 8.0, resuspended in the same buffer, and passed through a 5 ⁇ m 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 ⁇ l, to a final concentration of 50 ⁇ ).
  • the production of the second emulsion (water-in-oil-in-water) and sorting were performed as described. More than 10 6 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 ⁇ l 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 100 ⁇ g/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%.
  • FACSAria Becton-Dickinson
  • the pellets were resuspended in 200 ⁇ l of lysis buffer (0.1M Tris- HCl pH 8.0, ImM CaCl 2 , 10 ⁇ g/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.
  • 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 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 ⁇ ) and substrate concentrations was applied (from 0.3 x KM up to 2-3 x Km).
  • CMP-coumarin 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 3.7xl0 4 M -1 cm -1 ).
  • the half-life of the in situ generated GF in activity buffer (50 mM Tris, 1 mM CaCl 2 , 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).
  • 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.
  • GF kcat/ ⁇ values with in-situ generated GF.
  • the nominal racemic GF concentration was set to 40-50 nM.
  • 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 ⁇ l 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 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 50 ⁇ 1 diluted lysate were mixed with ⁇ of 6 ⁇ 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.
  • 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 (0.1-0.01 ⁇ ) 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.
  • mice 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 (50-75 ⁇ 1, retero-orbital) into heparin ( ⁇ ,1:10).
  • the targeted substitutions library was based on PONl's active-site structure.
  • 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.
  • the libraries comprised mostly inactive variants.
  • 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-Coumarin 12 .
  • 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 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).
  • 3D8 exhibited a k cat IK M value of 1.2xl0 7 M -1 rnin -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.
  • the concentration of the CMP- coumarin substrate was decreased to enable the isolation of variants with improved K M as well as k cat .
  • 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 ⁇ 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.
  • 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.
  • the toxicity of nerve agents prevents their use in ordinary labs.
  • 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 ( ⁇ 10 5 M -1 min -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 lxLD 5 o exposure (1 ⁇ ).
  • 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.
  • mice with atropine and 2-PAM had 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.
  • 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 samples 15 . 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).
  • variant 1G3 plate # 1, well G3.
  • variant 4D2 plate # 4, well D2.
  • Non-synonymous mutations observed in each variant Mutations in active site residues are noted by underline.
  • variant 2F8 plate # 2, well F8. .
  • Table 8 Activities of selected rePONl variants on Sp-CMP-coumarin and its a,b,c,d
  • CMP-F 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.
  • butyrylcholinesterase dose required for protection against organophosphates toxicity a mathematically based toxicokinetic model. Toxicol Sci 77, 358-67 (2004).
  • organophosphorus hydrolase variants with high degradation activity towards organophosphate pesticides Protein Eng Des Sel 19, 99-105 (2006).

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Abstract

Un polypeptide isolé selon l'invention comprend la séquence d'acides aminés de paraoxonase (PON1) sérique ayant une efficacité catalytique de k cat /K M ≈106 M-1min-1 pour un substrat d'agent nerveux.
EP10768078A 2009-09-17 2010-09-15 Polypeptides pon1 isolés, polynucléotides codant pour ceux-ci et utilisations de ceux-ci dans le traitement ou la prévention des dommages associés à l'exposition à un organophosphate Withdrawn EP2478093A2 (fr)

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