WO2013007570A1 - Procédé de criblage grande vitesse de l'activité lipase et/ou d'inhibiteurs de lipase dans des échantillons biologiques et des milieux de culture - Google Patents

Procédé de criblage grande vitesse de l'activité lipase et/ou d'inhibiteurs de lipase dans des échantillons biologiques et des milieux de culture Download PDF

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WO2013007570A1
WO2013007570A1 PCT/EP2012/063026 EP2012063026W WO2013007570A1 WO 2013007570 A1 WO2013007570 A1 WO 2013007570A1 EP 2012063026 W EP2012063026 W EP 2012063026W WO 2013007570 A1 WO2013007570 A1 WO 2013007570A1
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acid
lipase
sample
molecules
wells
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Jean-François CAVALIER
Frédéric CARRIERE
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Centre National De La Recherche Scientifique
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/61—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving triglycerides

Definitions

  • Lipases occur widely in the microbial (Jaeger et al, FEMS Microbiol. Rev. 1994, 15, 29- 63; Cotes et al, Applied Microbiol. Biotechnol. 2008, 78, 741-749), plant (Mukherjee, Prog. Lipid Res. 1994, 33, 165-174) and animal kingdoms (Carriere et al, J. Mol. Cat. B: Enzymatic 1997, 3, 55-64). These enzymes play an important role in fat digestion, lipoprotein metabolism, and in the mobilization of fat stored in lipid inclusion bodies, endosperms and adipocytes.
  • the catalytic process can be described as a reversible lipase adsorption/desorption step occurring at the oil/water interface, followed by the formation of an interfacial enzyme substrate complex and the release of lipolysis products (Verger et al., J. Biol. Chem. 1973, 248, 4023-4034).
  • pNP esters can also be hydro lysed by non enzymatic proteins, and consequently they should not be used to assay lipase activities, even with purified lipases.
  • TAGs are naturally fluorescent because more than half of the fatty acids from Parinari oil are known to contain parinaric acid (9,1 1 ,13, 15 -octadecatetraenoic acid) in its esterified form (Riley, J. Chem. Soc. 1950, 12-18).
  • this method requires the presence of a detergent in order to solubilize the released parinaric acid into mixed micelles.
  • This phase change (from an emulsified phase to a micellar phase) is accompanied by a variation of fluorescence spectral emission which has been exploited to monitor, with great sensitivity, the progress of the enzymatic reaction.
  • Pencreac'h et al. have developed a sensitive UV spectrophotometric lipase assay using less oxidation-sensitive TAGs extracted from Aleurites fordii seeds (Tung oil) (Pencreac'h et al., Anal. Biochem. 2002, 303, 17-24).
  • Crude Tung oil contains up to 72% a-eleostearic acid (9Z, l l£ ' , 13£-octadeca-9,l l,13-trienoc acid); an octadecatrienoic fatty acid esterified in the 1,3 position of the TAGs present in Tung oil (Radunz et al, Z. Naturaba 1998, 53, 305-310; Laguerre et al, Anal. Biochem. 2008, 380, 282-290).
  • the conjugated triene present in a-eleostearic acid constitutes an intrinsic chromophore, which confers strong UV absorption properties on both the free fatty acid (Sklar et al., Proc. Natl.
  • This lipase assay is based on the difference of the absorption coefficients between a-eleostearic ester on the triacylglycerol backbone and the free ⁇ -eleostearic acid resulting from the lipo lysis.
  • a change in the absorption spectrum UV is observed during the lipolysis and the enzymatic activity can be quantified continuously by measuring the optical density increase at 272 nm.
  • the prior application WO 2006/085009 describes the use of purified TAGs of Tung oil as well as synthetic prochiral or chiral esters of a-eleostearate (such as citronellol a-eleostearate) to coat the wells of microtitration plates (constituted by plastic material which is non-absorbent in the ultraviolet) with a very thin film (equivalent to a few hundreds of monomolecular layers).
  • a-eleostearate such as citronellol a-eleostearate
  • a major limitation of this technique is related to the fact that this technique only uses Tung (Aleurites fordii) oil TAGs as well as its derivatives (synthetic esters, triacylglycerols ...) containing ⁇ -eleostearic acid as substrate of a lipase activity. Indeed, some lipases of interest do not have significant lipolytic activity with coated Tung oil TAGs. Consequently, the technique described in WO 2006/085009 could have limited applications, in particular in the screening of unknown lipases from various sources or in in vitro therapeutic diagnostic.
  • Another drawback of this method is that this method uses hexane, a toxic evaporable solvent, which may cause impaired fertility and central nervous system depression in human being and is dangerous for environment.
  • microtiterplates were not resistant towards hexane.
  • hexane when used as solvent of lipid substrate for microtitration plate preparation, it can dissolve the plastic and lead to a significant alteration of the plastic material at the bottom of the well. Consequently, the presence of plastic resins can strongly alter lipase activity and UV adsorption of lipid substrate.
  • ethanol containing solvent can be used as solvent of lipid substrate in the preparation for microtitration plate coated by natural oils as well as their derivatives containing conjugated polyunsaturated fatty acids, without interfering microtitration plate wells or altering lipase activity measurement result.
  • the purpose of the present invention is to provide an improved method for preparing microtitration plates coated by a suitable substrate.
  • the purpose of the present invention is also to provide a microtitration plate coated by a suitable substrate for lipases.
  • Another purpose of the present invention is to provide a wide spectrum method for detecting or measuring in vitro a lipase activity and/or inhibition capacity of an inhibitor in a sample.
  • the purpose of the present invention is also to provide a method for screening lipolytic enzymes from various sources.
  • the purpose of the present invention is also to provide a method for screening inhibitors of lipolytic enzymes.
  • the purpose of the present invention is also to provide a method for directly screening the lipase mutants produced in recombinant form.
  • the present invention relates to a method for preparing microtitration plates comprising wells coated with a lipid substrate.
  • Said method comprises the following step:
  • said ethanol containing solvent does not contain hexane, petroleum ether, chlorinated solvents, or diethyl ether.
  • the solvent containing pure ethanol can contain other non-toxic alcohol having low viscosity and consequently high evaporation rate, such as n-propanol, isopropanol, n-butanol, isobutanol, sec-butanol, or teri-butanol, with the proviso that said solvent does not contain hexane, petroleum ether, chlorinated solvents, or diethyl ether.
  • other non-toxic alcohol having low viscosity and consequently high evaporation rate such as n-propanol, isopropanol, n-butanol, isobutanol, sec-butanol, or teri-butanol, with the proviso that said solvent does not contain hexane, petroleum ether, chlorinated solvents, or diethyl ether.
  • non-toxic alcohol we understand an alcohol which is classified as a Class 3 solvent according to the ICH Harmonised Tripartite Guideline Q3C(R5), February 2011.
  • lipid substrate refers to any molecule upon which a lipase acts.
  • a lipid substrate is often insoluble in water.
  • a lipase is an enzyme which hydrolyzes the ester chemical bonds in lipid substrates.
  • chlorinated solvents refers to organic compounds which can be used as solvent containing at least one covalently bonded chlorine atom in its molecule structure. Chlorinated solvents mean in particular methylene chloride (chloromethane family), perchloroethylene and trichloroethylene.
  • the pure ethanol used in the solvent for solubilising a lipid substrate can be a commercial ethanol having a concentration ranging between 95% to 99.9%, and preferentially 99.9%.
  • the steps (i) and (ii) of said method can be repeated in several cycles to coat layer by layer the substrate on the wells of microtitration plate. Each cycle comprises sequentially the step (i) and then the step (ii) of the method described above.
  • the lipid substrate is solubilized in the solvent with a concentration from 0.5 mg/mL to 1.5 mg/mL, preferably 1.0 mg mL.
  • the method for preparing microtitration plates includes a step of sonication of the lipid substrate before the step (i), which could help the lipid substrate to be better solubilized in solvent.
  • the steps (b) and (c) of said method can be repeated in several cycles to coat layer by layer the substrate on the wells of microtitration plate.
  • the lipid substrate used in the method as defined above is chosen from:
  • R l s R 2 and R 3 represent independently of each other:
  • a fatty acyl group represented by -COR in which R represents alkyl residues of identical or different conjugated fatty acids, comprising 12-20 carbon atoms, and optionally having one or more conjugated unsaturations,
  • Bossiella orgigniana containing 20:5 (n-6) bosseopentaenoic acid (5Z, 8Z, 10E, 12E, 14Z), or
  • esters of cholesterol, or of alcohols or of molecules which are prochiral or chiral and are of pharmaceutical interest such as citronelol, propanolol, sotalol or carvedilol, or of molecules of industrial interest such as menthol with a fatty acid issued from:
  • Punica granatum seed oil containing 18:3 (n-5) punicic acid (9Z, 11Z, 13E); - the red coralline algae, Bossiella orgigniana, containing 20:5 (n-6) bosseopentaenoic acid (5Z, 8Z, 10E, 12E, 14Z).
  • alcohols or of molecules which are prochiral refers to alcohols or molecules which are achiral, but can be converted from achiral to chiral in a single desymmetrization step, or alcohols or molecules which are achiral and contains a trigonal system and which can be made chiral by the addition to the trigonal system of a new atom or achiral group (IUPAC Compendium of Chemical Terminology 2nd Edition (1997)).
  • alcohols or of molecules which are chiral refers to alcohols or molecules which lack an internal plane of symmetry and thus has a non-superposable mirror image.
  • the invention relates to a method as defined above, wherein the lipid substrate is a triacylglycerols of formula I, wherein 3 ⁇ 4 and R 3 are as defined above and R2 represents a fatty acid acyl residue issued from:
  • Bossiella orgigniana containing 20:5 (n-6) bosseopentaenoic acid (5Z, 8Z, 10E, 12E, 14Z).
  • the invention relates to a method as defined above, wherein the lipid substrate is a triacylglycerols of formula I, wherein Ri and R 3 are as defined above and wherein R2 represents a fatty acid acyl residue issued from Punica granatum seed oil, containing 18:3 (n-5) punicic acid (9Z, 11Z, 13E).
  • the present invention concerns particularly an aforementioned method, wherein the lipid substrate is chosen from:
  • a fatty acyl group represented by -COR in which R represents alkyl residues of fatty acid issued of Punica granatum seed oil, containing 18:3 (n-5) punicic acid (9Z, 11Z, 13E) with the proviso that at least one of Rl, R2 and R3 is -COR, or
  • esters of cholesterol, or of alcohols or of molecules which are prochiral or chiral and are of pharmaceutical interest such as citronelol, propanolol, sotalol or carvedilol, or of molecules of industrial interest such as menthol with a fatty acid issued of Punica granatum seed oil, containing 18:3 (n-5) punicic acid (9Z, 11Z, 13E).
  • the invention relates to a method as defined above, wherein the lipid substrate is chosen from:
  • Rl, R2 and R3 represent independently of each other:
  • a fatty acyl group represented by -COR in which R represents alkyl residues of identical or different conjugated fatty acids, comprising 12-20 carbon atoms, and optionally having one or more conjugated unsaturations,
  • Rl, R2 and R3 is an acyl residues of a fatty acid chosen from:
  • esters of cholesterol, or of alcohols or of molecules which are prochiral or chiral and are of pharmaceutical interest such as citronelol, propanolol, sotalol or carvedilol, or of molecules of industrial interest such as menthol with a fatty acid chosen from:
  • rumenic acid (9Z, 1 IE), - 18:3 (n-6) a- calendic acid (8E, 10E, 12Z) or ⁇ -calendic acid (8E, 10E, 12Z)
  • the lipid substrate used in the method as defined above is a synthetic triacylglycerols of formula I, wherein Rj and R3 are as defined above and R2 represents an acyl residue of a fatty acid chosen from:
  • the invention relates to a method as defined above, wherein the lipid substrate is a synthetic triacylglycerols of formula I, wherein and R3 are as defined above and R2 represents the acyl residue of 18:3 (n-5) punicic acid (9Z, 11Z, 13E).
  • the invention relates to a method as defined above, wherein the lipid substrate is chosen from:
  • R l s R2 and R3 represent independently of each other:
  • the invention relates to a method as defined above, wherein the lipid substrate is natural purified TAGs from Punica granatum (Promegranate) seed oil.
  • Natural purified TAGs from Punica granatum seed oil contain up to 80% of punicate, 3% of palmitate, 2% of stearate, 5% of oleate, 5% of linoleate, and 5% of ester of other unsaturated fatty acids.
  • the present invention also relates to a microtitration plate comprising wells coated with a lipid substrate in a thickness from 0.5 to 5 ⁇ , said lipid substrate being chosen from
  • R l s R 2 and R3 represent independently of each other :
  • a fatty acyl group represented by -COR in which R represents alkyl residues of fatty acid issued of Punica granatum seed oil, containing 18:3 (n-5) punicic acid (9Z, 11Z, 13E) with the proviso that at least one of Ri, R2 and R3 is -COR, or
  • esters of cholesterol, or of alcohols or of molecules which are prochiral or chiral and are of pharmaceutical interest such as citronelol, propanolol, sotalol or carvedilol, or of molecules of industrial interest such as menthol with a fatty acid issued of Punica granatum seed oil, containing 18:3 (n-5) punicic acid (9Z, 11Z, 13E).
  • the present invention also relates to a method for screening inhibitors of lipolytic enzymes in pure buffer or in raw culture medium without further purification, characterized in that said method comprises the following step:
  • the present invention also relates to a method for screening directly at high flow rates the lipase mutants produced in recombinant forms in a sample in raw culture medium without any purification, characterized in that said method comprises the following step:
  • lipase activity we understand activity of any lipase directly isolated from mammal or microorganism (bacteria, fugi, etc.), or lipase obtained by genetic engineering, eventually modified to mutate, insert or delete one or more amino acids from a natural lipase, or any lipase synthesized by a chemical method known to one skilled in the art.
  • the lipase activity can be measured according to any conventional method well known in the art, in particular by the method described in particular in Beisson et al. (Eur. J. Lipid Sci. Techno 1. 2000, 2, 133-153).
  • inhibitor of lipolytic enzyme we understand any proteins, organic or inorganic, natural or synthetic molecules which can inhibit enzymatic activity of a lipase.
  • the present invention also relates to a method for detecting or measuring in vitro a lipase activity and/or inhibition capacity of a lipase inhibitor in a sample in aqueous solution, characterized in that said method comprises the following step:
  • the suitable sample for the detecting or measuring method as described above is a biological sample selected from the group comprising human or animal blood sample, gastrointestinal fluids, feces or homogenized biological tissues.
  • the suitable sample for the detecting or measuring method as described above could be also a purified sample and then dissolved in pure buffer or in raw culture medium without further purification.
  • the method according to the invention allows working directly on a raw culture medium without any further purification such as, for example, solvent extraction of the culture medium supernatant and organic phase concentration in vacuum for the screening of lipase inhibitors; concentration, gel filtration and/or column purification for the screening of a secreted lipase activity.
  • solvent extraction of the culture medium supernatant and organic phase concentration in vacuum for the screening of lipase inhibitors
  • concentration, gel filtration and/or column purification for the screening of a secreted lipase activity.
  • FIGs. 6a and 6b different tested culture media, compared to a pure buffer, do not have a significant impact on lipase assay sensitivity.
  • the lipase activity is measured by spectrophotometry in the UV absorption spectrum of the released conjugated fatty acid.
  • UV absorption spectrum of the released conjugated fatty acid can be carried out by any method known to one skilled in the art (see also the part "materials and methods" of the present application).
  • said detecting or measuring method comprises, prior to the step (i), a step wherein a buffer solution is constituted preferentially by Tris and containing ⁇ - cyclodextrin.
  • bile salts can be added to the wells of microtitration plates obtained according to the present invention.
  • ⁇ -cyclodextrin in this method is preferable in above described lipase activity measurement method.
  • the lipase binds to the lipid interface and starts hydrolyzing the coated TAGs, the released long carbon alkyl chains (> CI 6) free fatty acids are mainly insoluble and are often glued on the lipid interface.
  • the presence of ⁇ -cyclodextrin in the reaction buffer allows extracting, from the coated TAGs lipid phase, these free fatty acids released during hydrolysis of lipid substrate. This free fatty acid/ -cyclodextrin complex enables solubilising in buffer the free fatty acids formed (Laurent et al., Chem. Phys. Lipids 1994, 70, 35-42).
  • Bile salts are also sometimes necessary for maximize the enzymatic activity (Belle et al., Biochemistry 2007, 46, 2205-2214), as well as the inhibition rate (Moreau et al., Biochemistry 1991 , 30(4), 1037-1041 ; Cudrey et al, Biochemistry 1993, 32(50), 13800-13808; Ben Ali et al, Biochemistry 2004, 43(29), 9298-9306) of most lipases, including gastric and pancreatic lipases.
  • bile salts can induce and stabilize the active form of the protein, thus making the catalytic site accessible to the lipid substrate and/or the inhibitor (Belle et al., Biochemistry 2007, 46, 2205- 2214).
  • the buffer solution previously added to the wells of microtitration plates is at pH ranging from 4 to 9.
  • the present invention concerns also a method for in vitro diagnosing human or animal diseases linked to irregularity of plasma lipase level in said human or animal, said method comprising: - adding a biological sample obtained from said human or animal to the microtitration plates obtained according to the method as defined above,
  • the plasma lipase level obtained in said biological sample is compared with the plasma lipase level in a healthy individual.
  • pancreatic diseases such as acute pancreatitis, chronic pancreatitis, or renal failure
  • abdominal trauma such as ischemia, mesenteric infarct, intestinal perforation or occlusion, lysosomal acid lipase (LAL) deficiency, lipoprotein lipase deficiency.
  • LAL lysosomal acid lipase
  • FIG. 1 represents the operating condition protocol for the microtitration plate lipo lysis assay in a microtitration plate well coated by TAGs.
  • the release of the conjugated free fatty acids due to the lipolysis of the lipid substrate adsorbed in each well of the microtitration plates is monitored by U.V.
  • E represents lipase in solution
  • E* represents lipase adsorbed at the lipid interface
  • S represents TAG substrate
  • P represents lipolysis products (free fatty acids).
  • FIGs. 2a and 2b represents a lipase activity assay of porcine pancreatic lipase (PPL) contained in porcine pancreas extract (PPE) using respectively TAGs from Tung and Pomegranate oils which are coated on microtitration plates.
  • PPL porcine pancreatic lipase
  • PPE porcine pancreas extract
  • FIG. 2a represents the kinetics profile of PPL (5 ng PPE/well) for hydrolysis of purified TAGs respectively from Tung oil or Pomegranate oil.
  • the optical density (OD) of the released conjugated fatty acid is measured at 272 ran for Tung oil or at 275 ran for Pomegranate oil.
  • X-axis represents time (min);
  • Y-axis represents OD.
  • Samples of Pomegranate oil are represented by grey circle. Samples of Tung oil are represented by black diamond. Buffer alone is represented by open square. Time "0" means injection of PPL (5 ng contained in PPE) into the wells.
  • FIG. 2b represents the steady-state reaction rate of variable amounts of PPL for hydrolysis of purified TAGs respectively from Tung oil or Pomegranate oil.
  • 50 ⁇ ⁇ 11 of coated Tung or Pomegranate oil TAGs were incubated with variable amounts of PPL (contained in PPE) injected into the well containing 200 of buffer. Results are expressed as mean values of at least two assays (CV% ⁇ 5.0%).
  • X-axis represents lipase amount (ng PPL contained in PPE).
  • Y-axis represents reaction rate (AOD/min).
  • Samples of Pomegranate oil are represented by grey circle.
  • Samples of Tung oil are represented by black diamond.
  • FIGs. 3a and 3b represents LipY (triacylglycerol lipase Rv3097c from Mycobacterium tuberculosis) lipase activity assay, using TAGs from Tung and Pomegranate oils coated on microtiter plates.
  • LipY triacylglycerol lipase Rv3097c from Mycobacterium tuberculosis lipase activity assay, using TAGs from Tung and Pomegranate oils coated on microtiter plates.
  • FIG. 3a represents the kinetics profile of LipY (1.2 ⁇ ⁇ 1 ⁇ ) for hydrolysis of purified TAGs respectively from Tung oil or Pomegranate oil.
  • the optical density (OD) of the released conjugated fatty acid is measured at 272 ran for Tung oil or at 275 ran for Pomegranate oil.
  • X-axis represents time (min);
  • Y-axis represents OD.
  • Samples of Pomegranate oil are represented by grey circle. Samples of Tung oil are represented by black diamond. Buffer alone is represented by open square.
  • FIG. 3b represents the steady-state reaction rate of variable amounts of LipY for hydrolysis of purified TAGs from Pomegranate oil.
  • FIGs. 4a and 4b exhibit kinetics profile of PPL (5 ng contained in PPE) inhibited by Orlistat (Tetrahydrolipstatin).
  • the microplate is prepared by "coating" with TAGs (50 ⁇ 11) extracted from Pomegranate oil which contains a high proportion of punicic acid.
  • a solution (180 ⁇ ) of Tris buffer is added into the wells.
  • the activity of the PPL (5 ng contained in PPE) alone or pre- incubated 30 min. at 25°C with increasing molar fraction of Orlistat, is measured by recording the optical density increase at 275 nm, corresponding to the released of punicic acid.
  • FIG. 4a represents the increase of optical density (OD) at 275 nm in function of time.
  • FIG. 4b represents the effects of increasing Orlistat molar excess on the rate of lipo lysis of coated Pomegranate oil TAGs by Cutinase (O, 8 ng), GPLRP2 (Guinea Pig Lipase related protein 2; ⁇ , 5 ng), PPL (A, 5 ng contained in PPE) and LipY (triacylglycerol lipase Rv3097c from Mycobacterium tuberculosis; ⁇ , 0.6 ⁇ g). Each of these three enzymes was firstly pre-incubated at various enzyme/Orlistat molar ratios for 30 min at 25 °C. Results are expressed as mean values of at least two assays (CV% ⁇ 5.0%).
  • X-axis represents Orlistat to lipase molar excess; Y-axis represents lipase residual activity.
  • FIGs. 5a and 5b exhibit typical kinetic recordings of LipY inhibited by Orlistat (Tetrahydrolipstatin). LipY was incubated with Orlistat at an enzyme-inhibitor molar excess of 1 :20.
  • the curve with the black square corresponds to the lipase LipY alone; the curve with the grey circle corresponds to the lipase LipY pre-incubated with Orlistat after 1 min incubation period; the curve with the black triangle corresponds to the lipase LipY pre-incubated with Orlistat after 30 min incubation period; the curve with the open diamond corresponds to the lipase LipY pre-incubated with Orlistat after 60 min incubation period.
  • Kinetic recordings are representative of at least two independent experiments. The respective residual steady-state lipase activities are shown close to each kinetic curve.
  • a black arrow points the way of either the reactivation (FIG 5a) or the inhibition (FIG. 5b) process.
  • FIG. 5 ⁇ represents the hydrolysis of tributyrin emulsion by LipY, using the pH-stat technique. At various time intervals, samples of the incubation medium was injected in the pH-stat vessel, after recording the background hydrolysis for 2 min. Kinetic assays were performed in a thermostated (37°C) vessel containing 0.5 ml tributyrin emulsion mechanically emulsi ied in 14.5 ml of 2.5 mM Tris-HCl buffer (pH 7.5) containing 300 mM NaCl and 3 mM NaTDC. X-axis represents time (min); Y-axis represents the amount of free fatty acid released ⁇ mol).FIG.
  • 5b represents the increase of optical density (OD) at 275 nm corresponding to the hydrolysis of coated TAGs by LipY inhibited by Orlistat (Tetrahydrolipstatin).
  • X-axis represents time (min); Y-axis represents OD increase at 275 nm.
  • FIGs. 6a and 6b show the influence of various culture media on the enzymatic activity as well as on the inhibition of PPL (5 ng contained in PPE) by Orlistat.
  • FIG. 6a represents the relative values of AOD/min obtained when measuring on coated Pomegranate oil TAGs.
  • the lipolytic activity of PPL (5 ng contained in PPE) is prepared in each culture medium and then is diluted 3 Ox in the buffer prior to the assay.
  • Y-axis represents the relative reaction rate (AOD/min) compared to the one obtained in buffer (100%).
  • FIG. 6b represents the residual activity of PPL (5 ng contained in PPE) measured on coated Pomegranate oil TAGs.
  • the Orlistat solution used has been prepared in culture medium and then subjected to 30x dilution in DMSO.
  • Y-axis represents PPL residual activity.
  • FIG. 7 represents two microplate wells (on the left) coated by a lipid substrate dissolved in hexane as described in WO 2006/085009, and two microplates wells (on the right) coated by the same lipid substrate dissolved in ethanol as described in the present application.
  • TLC Thin-layer chromatography
  • TLC plates After being eluted, TLC plates were then dried, sprayed with copper acetate-85.5% phosphoric acid solution (50:50, v/v), and the neutral lipids were revealed by charring at 180°C for 10 to 15 min.
  • Triolein, diolein (1,2(2,3) and 1,3 isomers), monoololein and oleic acid were used as reference standards for the TLC analysis.
  • the polar compounds (diacylglycerols, free fatty acids, oxidation products, tocopherols) of the crude Tung or Pomegranate oil were removed by passing 25 mL of a 200 mg/mL oil solution prepared in petroleum ether 60/40 containing 0.01% (w/v) of Butylhydroxytoluene (BHT), followed by elution with petroleum ether 60/40 containing 0.01% (w/v) of BHT (eluent) through an alumina column prepared as follows: 30 g of aluminium oxide 90 basic activated in 50 mL of eluent was introduced into a glass column and the excess solvent was eliminated until it rose to the alumina surface.
  • BHT Butylhydroxytoluene
  • the total FFAs were extracted from the silica with diethyl ether containing 0.01% BHT. The amount of total FFA recovered was determined by measuring the constant dry weight after the solvent had evaporated. The purified FFAs were finally dissolved in ethanol containing 0.01% (w/v) BHT at 1.2 mg.mL "1 final concentration. This stock solution was stable (no changes were observed in the UV spectrum) for at least 1 month.
  • TAGs from Pomegranate oil containing up to ⁇ 70-80% punicic acid were dissolved in pure ethanol containing 0.01% (w/v) BHT to a final concentration of 1 mg/mL.
  • the obtained TAGs ethanolic solution was sonicated for 2x 25 min. at 25°C. 50 ⁇ L of this substrate solution was applied to each well of 96-well Costar ® UV-transparent microtiter plate and the solvent was evaporated under vacuum and in absence of light for nearly 180 min. After ethanol evaporation, the coated TAGs were found to be stable for at least 1 week at 4°C and in the absence of light.
  • the lipase assay was performed at 37°C in 10 mM Tris-HCl buffer (pH 8) containing 150 mM NaCl, 6 mM CaCl 2 , 1 mM EDTA, 0.001% (w/v) BHT and 3 mg/mL ⁇ -Cyclodextrin ( ⁇ -CD).
  • the lipase then binds to the lipid interface and starts hydrolyzing the coated TAGs thus releasing FFAs which were complexed by ⁇ -CD present in the buffer (Laurent et al., Chem. Phys. Lipids 1994, 70, 35-42).
  • the formation of the ⁇ -CD / free fatty acid complex was monitored at 272 nm or 275 nm, corresponding to the maximum UV absorbance of a-eleostearic acid or punicic acid in solution, respectively.
  • the optical density (OD) increase at the selected wavelength was then continuously recorded for 40 min. at regular time intervals of 30 s against the buffer alone, using a microtiter plate scanning spectrophotometer (PowerWaveTM, Bio-Tek Instruments).
  • the apparent molar extinction coefficient ( ⁇ ⁇ ) of a- eleostearic acid at 272 nm was estimated to be 6519 ⁇ 642 M _1 .cm _1 and the one for punicic acid at 275 nm was estimated to be 5964.2 + 298 M ⁇ .cm "1 .
  • Specific activities (SA) were here expressed in U per cm 2 of the coated-TAGs in the well microtitration plates and at a molar concentration of lipase.
  • the lipase-inhibitor pre-incubation method was used to test, in aqueous medium and in the absence of substrate, the direct interactions between lipases and inhibitors (Ransac et al., Methods Enzymol. 1997, 286, 190-231). 10 mg/mL (20 mM) stock solution of Orlistat in dimethyl sulfoxide (DMSO) was first prepared.
  • DMSO dimethyl sulfoxide
  • the following lipases stock solutions were used: 0.25 ⁇ g mL PPL contained in pancreatin powder in 10 mM MES (pH 7.0) containing 150 mM NaCl; 0.25 ⁇ g/mL GPLRP2 in 20 mM Tris (pH 8.0) containing 150 mM NaCl; 0.4 ⁇ g mL Cutinase in in 10 mM Acetate buffer (pH 6.0); 0.2 mg mL LipY in 10 mM Tris (pH 8.0) containing 150 mM NaCl. An aliquot of each of these above mentioned enzymes solutions was pre-incubated with each inhibitor at various lipase/inhibitor molar ratios for 30 min.
  • Ml Glucose 15 g/L; Soy Peptone 15 g/L; CaC0 3 2 g/L; NaCl 5 g/L; Yeast Extract 2 g/L.
  • M2 Glucose 5 g/L; Soy Peptone 20 g/L; Glycerol 20 g/L; NaCl 3 g/L; Yeast Extract 5 g/L;
  • M3 Glucose 10 g/L; Soy Peptone 4 g/L; MgS0 4 0.25 g/L; NaCl 5 g/L; Yeast Extract 2 g/L.
  • M4 LB 25 g/L; Glycerol 15 g/L; NaCl 2.5 g/L; Tween 80 1 g/L; Olive oil 10 g/L.
  • YP Yeast Extract 10 g/L; Peptone 20 g/L.
  • ⁇ YPD Difco® YPD medium, purchase from BD
  • microtitration plates of the present invention coated by lipid substrates chosen from molecules of industrial and/or pharmaceutical interest and in particular from triacylglycerols extracted from pomegranate oil containing conjugated fatty acids make the microtitration plates suitable for a more sensible wide range lipase assay.
  • microtitration plates obtained by the method described in the present application which uses ethanol as solvent, can give a more accurate lipase activity measurement results.
  • microtitration plates of the Invention can be used in the screening of inhibitors of lipolytic enzymes.
  • FIGs. 4a and 4b show that the inhibition capacity of a potential lipolitic enzyme inhibitor can be correlated with the residual activity of lipolitic enzyme tested by the microtitration plates of the present invention. 4. Microtitration plates without any phenomenon of lipase reactivation
  • the lipase reactivation phenomenon has already been described in the case of human pancreatic lipase (HPL) and Orlistat, using the titrimetic pH-stat technique (Tiss et al, J. Mol. Cat. B: Enzymatic 2009, 58, 41-47). It was indeed shown that the inhibition of HPL could be rapidly and partially reversed in the presence of an emulsion of short- or long-chain triacylglycerols, as indicated by a kinetic reactivation process. In addition, the presence of bile salts in the lipase assay was found to enhance this reactivation process probably by forming mixed micelles between bile salts and Orlistat, which accelerates the deacylation phenomenon.
  • LipY activity is measured on TAG emulsions using the pH-stat technique (FIG. 5a) or microtitration plate coated by Pomegranate oil (FIG. 5b). After few minutes of incubation with Orlistat, LipY activity measured on TAG emulsions using the pH-stat technique was drastically reduced. The LipY lipolytic activity, however, progressively increased with time and was practically restored (by around 84% after 60 min of incubation - FIG. 5a) reaching a steady- state regime. By contrast, when the same aliquots of inhibited LipY were analyzed using the microtiter plate lipase assay and Pomegranate oil as substrate, linear kinetics were obtained in each case without any visible reactivation process with time (FIG. 5b).
  • FIGs. 5a and 56 show that the microtitration plate of the present invention can more accurately measure a lipase activity in the presence of a lipase inhibitor.
  • microtitration plates of the present invention obtained according to the "coating" method can be used for the screening of lipolytic enzymes and/or lipase inhibitors directly from raw culture medium.
  • a dilution factor in the buffer between 20 and lOOx, and preferably 30x was applied to each of the culture media tested in order to obtain a minimum background noise, i. e. OD values similar to those of the buffer alone.
  • the average of the relative AOD/min of the culture medium wherein a quantity of PPL equal to 5 ng is diluted is 103.7% +6.1%, compared to 100.0% +5.0% when the same lipase is diluted in the buffer, as seen from FIG. 6a.
  • These relative OD values are of the same order of magnitude, regardless of the culture medium used (see FIG. 6a).
  • the average of PPL (5 ng contained in PPE) residual activity after inhibition by Orlistat is 32.6% ⁇ 1.7% in presence of culture medium compared to 33.1% ⁇ 1.1% in buffer (see FIG. 6b).
  • lipid substrates chosen from molecules of industrial and/or pharmaceutical interest; and in particular from the purified triacylglycerols from natural oil containing conjugated fatty acids;
  • This method has been adapted to the high-speed screening of lipases and/or lipase inhibitors by the direct assay of culture media.

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Abstract

Cette invention concerne un procédé amélioré de préparation de microplaques de titrage comprenant des puits tapissés d'un substrat lipidique. Elle concerne également un procédé de détection ou de mesure in vitro de l'activité lipase et/ou de la capacité inhibitrice d'un inhibiteur présent dans un échantillon.
PCT/EP2012/063026 2011-07-12 2012-07-04 Procédé de criblage grande vitesse de l'activité lipase et/ou d'inhibiteurs de lipase dans des échantillons biologiques et des milieux de culture WO2013007570A1 (fr)

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US10047112B2 (en) 2014-02-21 2018-08-14 The Curators Of The University Of Missouri Fluorescent labeled inhibitors

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US10047112B2 (en) 2014-02-21 2018-08-14 The Curators Of The University Of Missouri Fluorescent labeled inhibitors

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