WO2003062461A1 - Fmdv leader peptidase assay - Google Patents

Fmdv leader peptidase assay Download PDF

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WO2003062461A1
WO2003062461A1 PCT/GB2003/000164 GB0300164W WO03062461A1 WO 2003062461 A1 WO2003062461 A1 WO 2003062461A1 GB 0300164 W GB0300164 W GB 0300164W WO 03062461 A1 WO03062461 A1 WO 03062461A1
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μmol
general formula
protective surfactant
protective
surfactant
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PCT/GB2003/000164
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French (fr)
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Manoj Kumar Ramjee
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Amura Therpeutics Limited
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • 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
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase

Definitions

  • THIS INVENTION relates to novel activity assay conditioning media resulting in a significant improvement in foot-and-mouth disease virus (FMDV) leader proteinase/peptidase activity and uses thereof.
  • FMDV foot-and-mouth disease virus
  • Foot-and-mouth disease virus is a member of the picornavirus family and is a major human and animal pathogen.
  • At least three peptidases are encoded by the single open reading frame genome, which encompasses approximately 7000 nucleotide bases.
  • the three peptidases are the 3C protease (3C pr0 ), the 2A protease (2A pro ) and leader protease (L pro ) (Seipelt, J., et al, (1999), Virus Res, 62 (2), 159-168).
  • the 3C protease, a chymotrypsin-like serine peptidase is essential for viral replication since it carries out the majority of the poly-protein cleavages during viral maturation.
  • the 2 A protease a chymotrypsin-like peptidase of un-known catalytic mechanism (Ryan, M. D., et al, (1998), Handbook of Proteolytic Enzymes, London, Academic Press, 1598-1600), carries out the cleavage between the viral capsid protein and non- structural precursor proteins (i.e. between the C-terminus of VP1 and its own N- terminus) (Seipelt, J., et al, (1999), Virus Res, 62 (2), 159-168).
  • the leader protease carries out the cleavage between its C-terminus and the N-terminus of VP4 (Seipelt, J., et al, (1999), Virus Res, 62 (2), 159-168; Strebel, K. and Beck, E., (1986), J Virol, 58 (3), 893-899; Guarne, A., et al, (2000), JMol Biol, 302 (5), 1227-1240; Grubman, M. J., (1998), Handbook of Proteolytic Enzymes, London, Academic Press, 675-677).
  • leader protease shares little sequence identity with papain, it has been classified as such because of structural similarities it shares with papain (Guarne, A., et al, (2000), JMolBiol, 302 (5), 1227-1240; Guarne, A., et al, (1998), Embo J, 17 (24), 7469-7479; Guarne, A., et al, (1996), Protein Sci, 5 (9), 1931-1933). Since the leader protease is not essential for viral replication (Piccone, M. E., et al, (1995), J Virol, 69 (9), 5376-5382), its precise role in viral replication is a matter of debate.
  • leader peptidase may act to enhance viral replication by cleaving translation initiation factor eIF4G, thereby arresting host-cell translation (Kirchweger, R., et al, (1994), J Virol, 68 (9), 5677-5684).
  • a preliminary biochemical characterisation of leader protease reported a pH optimum around pH 8.5.
  • Peptidase activity could only be measured using a hexapeptide based in the C-terminus of the cleavage site of eIF4G (eIF4GI). Activity was salt-dependent, with activity decreasing as salt concentration increases (Guarne, A., et al, (2000), J Mol Biol, 302 (5), 1227-1240).
  • the assay employed relatively high concentrations of enzyme (2 ⁇ g/ml; 100 nM based on 19.8 kDa) and the hexapeptide substrate displayed relatively poor kinetics parameters (h K M -1.62 x 10 3 M ' V 1 ) (Guarne, A., et al, (2000), JMol Biol, 302 (5), 1227- 1240).
  • the current assay is therefore relatively insensitive. This insensitivity not only requires that relatively large amounts of enzyme and substrate were used in each assay, but also preferentially required more specialised instrumentation to maximise signal capture.
  • a method of assaying foot-and-mouth disease virus (FMDV) leader peptidase activity comprising assaying the peptidase activity in the presence of a protective surfactant.
  • FMDV foot-and-mouth disease virus
  • Protective surfactants are those which significantly increase the sensitivity of the assay and/or significantly stabilise peptidase activity in solution.
  • Such surfactants include:
  • R represents a mono- or di-saccharide residue and x is a number from 4 to 15;
  • examples of such surfactants include the following:
  • alkyl thioglycosides of the general formula:
  • R-S-(CH 2 ) X -CH 3 where R represents a mono- or di-saccharide residue and x is a number from 4 to 15;
  • examples of such surfactants include the following:
  • cyclohexyl-n-hexyl- ⁇ -D-maltoside examples include, but are not limited to, cyclohexyl-n-hexyl- ⁇ -D-maltoside; cyclohexyl-»-methyl- ⁇ -D-maltoside; n- decanoylsucrose; «-dodecanoylsucrose; n-decyl- ⁇ -D- maltopyranoside; n-decyl- ⁇ -D-thiomaltoside; n-octanoyl- ⁇ -D- glucosylamine; «-octyl- ⁇ -D-maltopyranoside; R-undecyl- ⁇ -D- maltopyranoside.
  • R represents a group capable of forming an anion, which may be paired with an appropriate cation, such as Na + ;
  • examples of such surfactants include the following:
  • n is a number from 5 to 15;
  • examples of such surfactants include the following: • When n equals 8 the compound is MEGA- 10;
  • n is a number from 5 to 15;
  • surfactants include the following:
  • n is a number from 5 to 15;
  • surfactants include the following:
  • surfactants include the following:
  • BRIJ ® 35 polyoxyethylene (23) lauryl ether; polyoxyethyleneglycol dodecyl ether.
  • surfactants include the following:
  • w is a number from 10 to 30, x is a number from 10 to 30, y is a number from 10 to 30, z is a number from 10 to 30 and R is an acyl group comtaining from 2-30 carbon atoms;
  • examples of such surfactants include the following:
  • the compound When w, x, y and z equal 20 and R equals C ⁇ H 23 CO 2 -(laurate) the compound is Tween ® 20 (polysorbate 20; polyoxyethylene(20)sorbitan monolaurate); • When w, x, y and z equal 20 and R equals C 17 H 3 CO 2 -(oleate) the compound is Tween ® 80 n-dodecyl-N,N-dimethylglycine;
  • surfactants include the following:
  • zwitterionic surfactants selected from amidosulphobetaine (ASB-14 and ASB-16); DDMAU (N-dodecyl-N,N-(dimethylammonio)undecanoate); and DDMAB (N-dodecyl-N,N-(dimethylammonio)butyrate);
  • dimethyl(C 5 -C 15 )alkylphosphine oxides such as APO-8; APO-9; APO- 10 (dimethyldecylphosphine oxide); APO-11 and APO-12 (dimethyldodecylphosphine oxide, whose structure is shown below).
  • SDS sodium n-dodecyl sulphate
  • alkyl glucosides such as Elugent
  • NDSB non-detergent sulphobetaines
  • Mixtures of two or more surfactants may be used.
  • Protective surfactants are used at a concentration which gives rise to the protective effect but which does not materially adversely affect the reaction catalysed by the enzyme.
  • concentration is that at which the reaction is optimally enhanced.
  • the peptidase activity may be assayed by determining the rate of conversion of substrate to product. Any convenient substrate may be used. Labelled substrates are preferred, especially those in which some detectable change in the label occurs upon cleavage. Substrates labelled with a FRET pair are especially suitable, as are other fluorescent labels which are quenched either before, or in principle after, cleavage; a preferred FRET pair labelled substrate is Abz-Arg-Lys-Leu-Lys-Gly-Ala-Gly-Ser- Tyr(NO 2 )-As ⁇ -NH 2 . Others include:
  • Abz and Tyr(3-NO 2 ) can be replaced by any FRET pair described in the prior art (Haugland, R. P., (2002), Handbook of fluorescent probes and research chemicals, Molecular Probes, Inc., Eugene, Oregon, USA; Wu, P. and Brand, L., (1994), Anal Biochem, 218(1), 1-13).
  • the peptidase activity may be assayed in the presence of a candidate modulator (enhancer or inhibitor) of peptidase activity, in which case the assay functions as a screen of such candidate compounds.
  • Inhibitors of FMDV leader peptidase may have valuable pharmacological properties.
  • Protective surfactants useful in the invention may also stabilise a preparation of FMDV leader peptidase.
  • a preparation of FMDV leader peptidase including a protective surfactant may be provided.
  • the preparation will generally be aqueous.
  • Figure 1 shows the dependence of FMDV leader protease peptidase activity as a function of alkyl glucosides detergent concentration
  • Figure 2 shows the dependence of FMDV leader protease peptidase activity as a function of n-dodecyl- ⁇ -D-maltoside detergent concentration
  • Figure 3 shows the dependence of FMDV leader protease peptidase activity as a function of cholate and deoxycholate detergent concentration
  • Figure 4 shows the dependence of FMDV leader protease peptidase activity as a function of Big CHAP detergent concentration
  • Figure 5 shows the dependence of FMDV leader protease peptidase activity as a function of Triton X- 100 detergent concentration
  • Figure 6 shows the dependence of FMDV leader protease peptidase activity as a function of NP-40 detergent concentration
  • Figure 7 shows the dependence of FMDV leader protease peptidase activity as a function of Brij 35 and Genapol C-100 detergent concentration
  • Figure 8 shows the dependence of FMDV leader protease peptidase activity as a function of Pluronic F-127 detergent concentration
  • Figure 9 shows the dependence of FMDV leader protease peptidase activity as a function of Tween 20 detergent concentration
  • Figure 10 shows the dependence of FMDV leader protease peptidase activity as a function of Empigen BB detergent concentration
  • Figure 11 shows the dependence of FMDV leader protease peptidase activity as a function of Zwittergent detergent concentration
  • Figure 12 shows the dependence of FMDV leader protease peptidase activity as a function of CHAPS and CHAPSO detergent concentration
  • Figure 13 shows the dependence of FMDV leader protease peptidase activity as a function of APO-12 detergent concentration
  • Figure 14 shows the dependence of FMDV leader protease peptidase activity as a function of SDS detergent concentration
  • Figure 15 shows the dependence of FMDV leader protease peptidase activity as a function of Elugent detergent concentration
  • Figure 16 shows the addition of 0.5 mM deoxycholate to assay buffer prevents the loss of FMDV leader protease peptidase activity during freeze-thaw cycles.
  • HPLC-MS analysis was performed on Agilent 1100 series LC/MSD, using automated Agilent HPLC systems (HP 1100 system; Agilent Technologies, Bracknell, UK), with a gradient of 10 - 90% B in A over 10 min. on Phenomenex Columbus C 8 , 5 ⁇ , 300A, 50 x 2.0 mm at 0.4 ml/min.
  • the mass spectrometer was set to API-ES ionisation mode, positive polarity; scanning in the 100-1500 Da mass range with a gas temperature was set to 350°C.
  • Semi-preparative HPLC purification of crude samples was performed on Phenomenex Jupiter C4 (5 ⁇ , 300 A) using a linear increasing gradient of solvent B in solvent A using the gradient indicated, at 4.0 ml/min. The eluant absorbance was monitored at 215 nm and fractions collected manually, then lyophilised.
  • FRET-based substrates fluorescence resonance energy transfer methodology
  • Peptide synthesis was carried out in 5 ml polypropylene plastic syringes fitted with an end-cap, a TEFLON scinter and stopper.
  • Resin (NOVASYN TGR resin; 0.2 mmol/g; CNBiosciences, Beeston, Nottingahmshire, U.K) was added to the syringe as required.
  • Resin was solvated in dimethylformamide (DMF) (3 -5ml) on a rotating bed (SRT1, Stuart Scientific; Fisher Scientific, Loughborough, Leicestershire, U.K) for approximately 10 min.
  • DMF dimethylformamide
  • Peptide synthesis was carried out in repetitive cycles consisting of a coupling step, a reagent wash step, an Fmoc de-protection step, then a de-protection wash step followed by the next coupling round. Between each step excess reagent and solvent were removed by application of a vacuum. Each coupling step was commenced by activating a three-mole excess of Fmoc-amino acid (with respect to total resin loading capacity) via 2-(lH-benzotriazole-l-yl)-l, 1,3,3- tetramethyluronium hexafluorophosphate (HBTU), 1-hydroxybenzotriazole (HOBt) and N-methylmorpholine (NMM).
  • HBTU 2-(lH-benzotriazole-l-yl)-l, 1,3,3- tetramethyluronium hexafluorophosphate
  • HOBt 1-hydroxybenzotriazole
  • NMM N-methylmorpholine
  • the activation mixture was pre-mixed in DMF (2- 3 ml) for 30 s and the coupling step initiated by addition to the drained resin.
  • the syringe was capped and agitated on the rotating bed for 1 h.
  • the excess reagents were then drained (by application of the vacuum) and the bed washed with 6 x DMF (5 ml per wash).
  • Fmoc deprotection then commenced by continuously washing the resin bed, under gravity flow, for 10 min. with piperidine:DMF (20%:80%).
  • the excess reagents were again drained (by application of the vacuum) and the bed washed with 10 x DMF (5 ml per wash).
  • the resin was drained as before and ready for the next round of coupling.
  • the crude products were precipitated by addition of 50 ml cold methyl tert-butyl ether (MTBE) and precipitates collected by centrifugation (5500 r.p.m. for 5 min). The supernatant was discarded and the process repeated.
  • the final crude products were re-dissolved in 50:50% acetonitrile:water and analysed by RP-HPLC-MS. Crude products at >97% purity by UV analysis, were subsequently lyophilised. When required, poorer quality crude products were purified by semi-preparative HPLC and desired fractions pooled then lyophilised.
  • the sixth coupling mixture consisted of Fmoc-(FmocHmb)-Gly-Opfp (61.7 mg, 75 ⁇ mol) and HOBt (11.5 mg, 75 ⁇ mol), coupling for approximately 8 h at room temperature.
  • the seventh coupling mixture consisted of Fmoc-Lys(Boc)-OH (35.1 mg, 75 ⁇ mol), HBTU (28.4 mg, 75 ⁇ mol), HOBt (11.5 mg, 75 ⁇ mol) and NMM (6.6 ⁇ l, 75 ⁇ mol), coupling for approximately 16 h at room temperature.
  • Product (4) was cleaved and purified by semi-preparative HPLC using the following column elution profile: 0-1 min., 12% buffer B; 1-15 min., 12-50% buffer B; 15-17 min., 50-90% buffer B; 17-19 min., 90% buffer B; 19-21 min., 90-12% buffer B; 21- 25 min., 12% buffer B.
  • the eluant absorbance was monitored at 230 nm and fractions were collected manually.
  • Product containing fractions were lyophilised to give (4), yield 3.2 mg (2.5 ⁇ mol, 10%), ESI-MS 419.9 [M + 3H] 3+ , 629.2 [M + 2H] 2+ (calc. Mw 1256.4) with Rt 4.2 min (>97%).
  • the sixth coupling mixture consisted of Fmoc- (FmocHmb)-Gly-O ⁇ f ⁇ (61.7 mg, 75 ⁇ mol) and HOBt (11.5 mg, 75 ⁇ mol), coupling for approximately 8 h at room temperature.
  • the seventh coupling mixture consisted of Fmoc-Lys(Boc)-OH (35.1 mg, 75 ⁇ mol), HBTU (28.4 mg, 75 ⁇ mol), HOBt (11.5 mg, 75 ⁇ mol) and NMM (6.6 ⁇ l, 75 ⁇ mol), coupling for approximately 16h at room temperature.
  • Product (5) was cleaved and purified by semi-preparative HPLC using the following column elution profile: 0-1 min., 12% buffer B; 1-15 min., 12-50% buffer B; 15-17 min., 50-90% buffer B; 17-19 min., 90% buffer B; 19-21 min., 90-12% buffer B; 21- 25 min., 12% buffer B.
  • the eluant absorbance was monitored at 230 nm and fractions were collected manually.
  • Product containing fractions were lyophilised to give (5), yield 3.2 mg (2.5 ⁇ mol, 10%), ESI-MS 428.9 [M + 3H] 3+ , 642.7 [M + 2H] 2+ , 1284.3 [M + H] + (calc. Mw 1283.16) with Rt 4.2 min (>95 %).
  • Product (6) was cleaved (90 min) and purified by semi-preparative HPLC using the following column elution profile: 0-10 min., 10-90% buffer B; 10-12.5 min., 90% buffer B; 12.5 min., 10% buffer B. The eluant absorbance was monitored at 230 nm and fractions were collected manually. Product containing fractions were lyophilised to give, yield 4.4 mg (3.0 ⁇ mol, 12%), ESI-MS 490.0 [M + 3H] 3+ , 734.3 [M + 2H] 2+ (calc. Mw 1466.66) with Rt 3.9 min (>97%).
  • Substrate (7) was cleaved and lyophilised, yield 3.2 mg (2.5 ⁇ mol, 10%), ESI-MS 626.7 [M + 2H] 2+ , 1252.2 [M + H] + (calc. Mw 1251.36) with Rt 5.4 min (>95 %).
  • Fluorescence high throughput assays were carried out in either 384-well microtitre plates (Corning COSTAR 3705 plates, Fisher Scientific) or 96-well 'U' bottomed MICROFLUOR Wl microtitre plates (Thermo Labsystems, Ashford, Middlesex, U.K). Fluorescence assays were monitored using a SPECTRAMAX Gemini fluorescence plate reader (Molecular Devices).
  • HEPES Hydroxyethyl- piperazine ethanesulphonate
  • tris-hydroxylmethylaminomethane (tris) base bis-tris-propane
  • BTP bis-tris-propane
  • all the biological detergents e.g. TRITON X-100, TWEEN 20, CHAPS, ⁇ -octyl-gluopyranoside; zwittergents, etc.
  • TRITON X-100, TWEEN 20, CHAPS, ⁇ -octyl-gluopyranoside; zwittergents, etc. were purchased from CN Biosciences UK, Beeston, Nottinghamshire, U.K. Where applicable, the commercial name and published biophysical properties of each of the detergents were used (Bhairi, S.
  • Recombinant wild-type FMDV-LP was obtained from Dr. Tim Skern (Institut fur Medizinische Biochemie, Abiller fur Biochemie, Universtat Wien, Vienna, Austria). Stock solutions (10% (w/v)) of each detergent were prepared in purified water. Aliquots (5 ⁇ l) of each detergent were added to wells of row A of a 384 well microplate followed by 25 ⁇ l water to rows B to P of the same plate and a further 45 ⁇ l to row A. Row A was mixed thoroughly and 25 ⁇ l transferred to row B. The procedure was continued down the plate to produce a double dilution series down the plate from row A to row O; row P left as minus detergent controls.
  • FMDV-LP 40 nM
  • 50 mM tris-acetate, pH 8.4, 1 mM EDTA and 10 mM L-cysteine 50 mM tris-acetate, pH 8.4, 1 mM EDTA and 10 mM L-cysteine.
  • An aliquot (12 ⁇ l) of this enzyme stock was added to each well using a multi-channel pipette, starting at row P and working up the plate.
  • a stock solution (200 ⁇ M) of substrate (Abz-Arg-Lys-Leu-Lys-Gly-Ala- Gly-Ser-Tyr(NO 2 )-Asp-NH 2 ; K M a p ⁇ 51 ⁇ M; Incenta Limited) was prepared in 50 mM tris-acetate, pH 8.4, 1 mM EDTA, 10 mM L-cysteine. An aliquot (12 ⁇ l) of this stock was also added to each well using a multi-channel pipette, starting at row P and working up the plate. The rate of conversion of substrate to product was derived from the slope of the increase in fluorescence monitored continuously over time at 25 ⁇ 1°C.
  • Zwittergent ® is a registered trademark of Calbiochem-Novabiochem Corporation
  • Triton ® is a registered trademark of Rohm and Haas Company
  • BRIJ ® is a registered trademark of ICI Americas Incorporated
  • EMPIGEN ® is a registered trademark of Albright & Wilson Limited
  • Genapol ® is a registered trademark of Hoescht AG
  • PLURONIC ® is a registered trademark of BASF Corporation
  • ELUGENTTM is a registered trademark of Calbiochem-Novabiochem Corporation

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Abstract

Foot-and-mouth disease virus (FMDV) leader peptidase activity is assayed in the presence of a protective surfactant. Such surfactants significantly enhance peptidase activity. This increase in activity results in increased sensitivity and means that less enzyme is used per assay. This increase in sensitivity also makes the assay amenable to adaptation for generic instrumentation (e.g. microtitre plates and readers). Suitable protective surfactants include (thio)alkyl glycosides, bile acids, glucamide polyoxyethylenes, n-dodecyl-N,N-dimethylglycine, zwitterionic detergents, dimethyl(C5-C15)alkylphosphine oxides, sodium n-dodecyl sulphate, alkyl glucosides, 3-([4-tert-octyl]phenoxy)-1-propane sulphonic acid sodium salt and non-detergent sulphobetaines.

Description

FMDV LEADER PEPTIDASE ASSAY
THIS INVENTION relates to novel activity assay conditioning media resulting in a significant improvement in foot-and-mouth disease virus (FMDV) leader proteinase/peptidase activity and uses thereof.
Foot-and-mouth disease virus is a member of the picornavirus family and is a major human and animal pathogen. At least three peptidases are encoded by the single open reading frame genome, which encompasses approximately 7000 nucleotide bases. The three peptidases are the 3C protease (3Cpr0), the 2A protease (2Apro) and leader protease (Lpro) (Seipelt, J., et al, (1999), Virus Res, 62 (2), 159-168). The 3C protease, a chymotrypsin-like serine peptidase, is essential for viral replication since it carries out the majority of the poly-protein cleavages during viral maturation. The 2 A protease, a chymotrypsin-like peptidase of un-known catalytic mechanism (Ryan, M. D., et al, (1998), Handbook of Proteolytic Enzymes, London, Academic Press, 1598-1600), carries out the cleavage between the viral capsid protein and non- structural precursor proteins (i.e. between the C-terminus of VP1 and its own N- terminus) (Seipelt, J., et al, (1999), Virus Res, 62 (2), 159-168). The leader protease carries out the cleavage between its C-terminus and the N-terminus of VP4 (Seipelt, J., et al, (1999), Virus Res, 62 (2), 159-168; Strebel, K. and Beck, E., (1986), J Virol, 58 (3), 893-899; Guarne, A., et al, (2000), JMol Biol, 302 (5), 1227-1240; Grubman, M. J., (1998), Handbook of Proteolytic Enzymes, London, Academic Press, 675-677). Although the leader protease shares little sequence identity with papain, it has been classified as such because of structural similarities it shares with papain (Guarne, A., et al, (2000), JMolBiol, 302 (5), 1227-1240; Guarne, A., et al, (1998), Embo J, 17 (24), 7469-7479; Guarne, A., et al, (1996), Protein Sci, 5 (9), 1931-1933). Since the leader protease is not essential for viral replication (Piccone, M. E., et al, (1995), J Virol, 69 (9), 5376-5382), its precise role in viral replication is a matter of debate. Nevertheless it has been suggested that the leader peptidase may act to enhance viral replication by cleaving translation initiation factor eIF4G, thereby arresting host-cell translation (Kirchweger, R., et al, (1994), J Virol, 68 (9), 5677-5684). A preliminary biochemical characterisation of leader protease reported a pH optimum around pH 8.5. Peptidase activity could only be measured using a hexapeptide based in the C-terminus of the cleavage site of eIF4G (eIF4GI). Activity was salt-dependent, with activity decreasing as salt concentration increases (Guarne, A., et al, (2000), J Mol Biol, 302 (5), 1227-1240). However, the assay employed relatively high concentrations of enzyme (2 μg/ml; 100 nM based on 19.8 kDa) and the hexapeptide substrate displayed relatively poor kinetics parameters (h KM -1.62 x 103 M'V1) (Guarne, A., et al, (2000), JMol Biol, 302 (5), 1227- 1240). The current assay is therefore relatively insensitive. This insensitivity not only requires that relatively large amounts of enzyme and substrate were used in each assay, but also preferentially required more specialised instrumentation to maximise signal capture. The requirement for larger amounts of material restricts the number of assays that can be undertaken for a given amount of material and more importantly hampers the screening of large libraries of compounds in the search for potential inhibitors. Although this insensitivity may be overcome by incubation of the enzyme reaction for an extended period, it is known to those skilled in the art that proteins, and in particular peptidases, loose activity over time. As such, this measure would have limited success based on empirical determination of the precise reaction conditions required to preserve peptidase activity.
Although the biochemical characterisation of the leader protease activity has been reported (Guarne, A., et al, (2000), JMolBiol, 302 (5), 1227-1240), however to our surprise, we have found that the addition of one or more protective surfactants to the assay buffer significantly enhanced peptidase activity. This increase in activity results in increased sensitivity and means that less enzyme is used per assay. This increase in sensitivity also makes the assay amenable to adaptation for generic instrumentation (e.g. microtitre plates and readers). The addition of protective surfactants helps to stabilise peptidase activity in solution and as such preserve leader protease activity for an extended period. According to a first aspect of the invention therefore, there is provided a method of assaying foot-and-mouth disease virus (FMDV) leader peptidase activity, the method comprising assaying the peptidase activity in the presence of a protective surfactant.
Protective surfactants are those which significantly increase the sensitivity of the assay and/or significantly stabilise peptidase activity in solution. Such surfactants include:
alkyl glycosides of the general formula
R-O-(CH2)x-CH3
where R represents a mono- or di-saccharide residue and x is a number from 4 to 15; examples of such surfactants include the following:
• When R is glucose and x equals 8 the compound is n-nonyl-β-D- glucopyranoside;
• When R is glucose and x equals 7 the compound is «-octyl-β-D- glucopyranoside; • When R is glucose and x equals 6 the compound is «-heptyl-β-D- glucopyranoside;
• When R is glucose and x equals 5 the compound is π-hexyl-β-D- glucopyranoside;
• When R is maltose and x equals 11 the compound is n-dodecyl-β- D-maltoside;
• When R is maltose and x equals 9 the compound is «-decyl-β-D- maltoside;
alkyl thioglycosides of the general formula:
R-S-(CH2)X-CH3 where R represents a mono- or di-saccharide residue and x is a number from 4 to 15; examples of such surfactants include the following:
When R is glucose and x equals 8 the compound is «-nonyl-β-D- thioglucopyranoside;
When R is glucose and x equals 7 the compound is «-octyl-β-D- thioglucopyranoside;
When R is glucose and x equals 6 the compound is «-heptyl-β-D- thioglucopyranoside;
When R is glucose and x equals 5 the compound is n-hexyl-β-D- thioglucopyranoside;
When R is maltose and x equals 11 the compound is «-dodecyl-β-
D-thiomaltoside;
When R is maltose and x equals 9 the compound is «-decyl-β-D- thiomaltoside
Other examples include, but are not limited to, cyclohexyl-n-hexyl- β-D-maltoside; cyclohexyl-»-methyl-β-D-maltoside; n- decanoylsucrose; «-dodecanoylsucrose; n-decyl-β-D- maltopyranoside; n-decyl-β-D-thiomaltoside; n-octanoyl-β-D- glucosylamine; «-octyl-β-D-maltopyranoside; R-undecyl-β-D- maltopyranoside.
bile acids of the general formula:
Figure imgf000005_0001
where R represents a group capable of forming an anion, which may be paired with an appropriate cation, such as Na+; examples of such surfactants include the following:
When R is O" and X equals H the compound is sodium deoxycholate (3α, 12α-dihydroxy-5β-cholanic acid sodium salt) or other corresponding salt or free acid;
When R is NHCH2CH2SO3 " and X equals H the compound is sodium taurodeoxycholate or other corresponding salt or free acid;
When R is NHCH2CO2 " and X equals H the compound is sodium glycodeoxycholate or other corresponding salt or free acid;
When R is O" and X equals OH the compound is sodium cholate
(3α, 7α, 12α-trihydroxy-5β-cholanic acid sodium salt) or other corresponding salt or free acid;
When R is NHCH2CH2SO3 " and X equals OH the compound is sodium taurocholate or other corresponding salt or free acid;
When R is NHCH2CO2 " and X equals OH the compound is sodium glycocholate;
(other examples include, but are not limited to, chenodeoxycholic acid; taurodehydrocholate; taurolithocholic acid; tauroursodeoxy- cholic acid and their salts;
glucamides of the general formula:
Figure imgf000006_0001
where n is a number from 5 to 15; examples of such surfactants include the following: • When n equals 8 the compound is MEGA- 10;
• When n equals 7 the compound is MEGA-9;
• When n equals 6 the compound is MEGA- 8;
glucamides of the general formula:
Figure imgf000007_0001
• When X equals H the compound is deoxy Big CHAP;
• When X equals OH the compound is Big CHAP (N,~N-bis-(3- gluconamidopropyl)cholamide)
polyoxyethylenes of the general formula
Figure imgf000007_0002
where n is a number from 5 to 15; examples of such surfactants include the following:
When n equals 9-10 the compound is reduced Triton® X-100 (reduced polyethylene glycol-p-isooctylphenyl ether); • When n equals 7-8 the compound is reduced Triton " X-l 14
polyoxyethylenes of the general formula
Figure imgf000008_0001
where n is a number from 5 to 15; examples of such surfactants include the following:
• When n equals 9-10 the compound is Triton® X-100, NP-40 (polyethylene glycol-/j-isooctylphenyl ether)
• When n equals 7-8 the compound is Triton® X-l 14
• polyoxyethylenes of the general formula
CH3(CH2)y-O(CH2CH2O)xH
where x is a number from 3 to 30 and y is a number from 5 to 15; examples of such surfactants include the following:
• When y equals 12 and x equals 8 the compound is Genapol X-80; • When y equals 12 and x equals 10 the compound is Genapol X-
100; . When y equals 9 and x equals 8 the compound is C10E8
(octaethyleneglycol mono-n-decyl ether); . When y equals 11 and x equals 5 the compound is C12E5 (pentaethyleneglycol mono-n-dodecyl ether);
• When y equals 11 and x equals 8 the compound is C12E8 (octaethyleneglycol mono-«-dodecyl ether);
• When y equals 11 and x equals 9 the compound is C12E9 (nonaethyleneglycol mono-«-dodecyl ether) (THESIT; ATLAS G2127; LUBROL PX); When y equals 11 and x equals 10 the compound is Genapol® C-100;
When y equals 11 and x equals 23 the compound is BRIJ® 35 (polyoxyethylene (23) lauryl ether; polyoxyethyleneglycol dodecyl ether).
polyoxyethylenes of the general formula
HO(CH2CH2O)x-(CH(CH3)-CH2O)y-(CH2CH2O)z-H
where x is a number from 50 to 150 and y is a number from 30 to 100 and z is a number from 50 to 150; examples of such surfactants include the following:
• When x equals 98, y equals 67 and z equals 98 the compound is
PLURONIC® F-127 (Poloxamer 407);
polyoxyethylenes of the general formula
Figure imgf000009_0001
where w is a number from 10 to 30, x is a number from 10 to 30, y is a number from 10 to 30, z is a number from 10 to 30 and R is an acyl group comtaining from 2-30 carbon atoms; examples of such surfactants include the following:
When w, x, y and z equal 20 and R equals CπH23CO2-(laurate) the compound is Tween® 20 (polysorbate 20; polyoxyethylene(20)sorbitan monolaurate); • When w, x, y and z equal 20 and R equals C17H3 CO2-(oleate) the compound is Tween® 80 n-dodecyl-N,N-dimethylglycine;
zwitterionic detergents of the general formula:
Figure imgf000010_0001
where x is a number from 5 to 25; examples of such surfactants include the following:
When x equals 7 the compound is Zwittergent® 3-08; When x equals 9 the compound is Zwittergent® 3-10; When x equals 11 the compound is Zwittergent® 3-12; When x equals 13 the compound is Zwittergent® 3-14; When x equals 15 the compound is Zwittergent® 3-16;
zwitterionic detergents of the general formula:
Figure imgf000010_0002
When R equals H the compound is CHAPS (3-[(3- cholamidopropyl)dimethylammino] - 1 -propanesulphonate) ; • When R equals OH the compound is CHAPSO (3-[(3- cholamidopropyl)dimethylammino] -2-hydroxy- 1 - propanesulphonate);
zwitterionic surfactants selected from amidosulphobetaine (ASB-14 and ASB-16); DDMAU (N-dodecyl-N,N-(dimethylammonio)undecanoate); and DDMAB (N-dodecyl-N,N-(dimethylammonio)butyrate);
dimethyl(C5-C15)alkylphosphine oxides, such as APO-8; APO-9; APO- 10 (dimethyldecylphosphine oxide); APO-11 and APO-12 (dimethyldodecylphosphine oxide, whose structure is shown below).
Figure imgf000011_0001
sodium n-dodecyl sulphate (SDS);
a mixture of alkyl glucosides such as Elugent;
• TOPPS ({3-([4-tert-octyl]phenoxy)-l-propane sulphonic acid sodium salt);
• non-detergent sulphobetaines (NDSB; see structures below).
Figure imgf000011_0002
NDSB-195
Figure imgf000011_0003
Figure imgf000012_0001
Mixtures of two or more surfactants may be used.
Protective surfactants are used at a concentration which gives rise to the protective effect but which does not materially adversely affect the reaction catalysed by the enzyme. Preferably the concentration is that at which the reaction is optimally enhanced.
The peptidase activity may be assayed by determining the rate of conversion of substrate to product. Any convenient substrate may be used. Labelled substrates are preferred, especially those in which some detectable change in the label occurs upon cleavage. Substrates labelled with a FRET pair are especially suitable, as are other fluorescent labels which are quenched either before, or in principle after, cleavage; a preferred FRET pair labelled substrate is Abz-Arg-Lys-Leu-Lys-Gly-Ala-Gly-Ser- Tyr(NO2)-Asρ-NH2. Others include:
Abz-Lys-Leu-Cys(Bzl)-Phe-Ser-Lys-Gln-Leu-Tyr(3-NO2)-Asp-NH2,
Abz-Leu-Val-Gly-Arg-Ala-Gln-Tyr(3-NO2)-Asp-NH2;
Abz-Lys-Leu-Lys-Gly-Ala-Gly-Tyr(3-NO2)-Asp-NH2;
Abz-Arg-Lys-Leu-Lys-Gly-Ala-Gly-Asn-Tyr(3-NO2)-Asp-NH2;
Abz-Ser-Val-Pro-Lys-Arg-Arg-Arg-Lys-Tyr(3-NO2)-Asp-NH2; and
Abz-Ala-Asn-Leu-Gly-Arg-Pro-Ala-Leu-Tyr(3-NO2)-Asp-NH2.
Abz and Tyr(3-NO2) can be replaced by any FRET pair described in the prior art (Haugland, R. P., (2002), Handbook of fluorescent probes and research chemicals, Molecular Probes, Inc., Eugene, Oregon, USA; Wu, P. and Brand, L., (1994), Anal Biochem, 218(1), 1-13). The peptidase activity may be assayed in the presence of a candidate modulator (enhancer or inhibitor) of peptidase activity, in which case the assay functions as a screen of such candidate compounds. Inhibitors of FMDV leader peptidase may have valuable pharmacological properties.
Protective surfactants useful in the invention may also stabilise a preparation of FMDV leader peptidase. According to a second aspect of the invention, there is therefore provided a preparation of FMDV leader peptidase including a protective surfactant. The preparation will generally be aqueous.
Preferred features of each aspect of the invention are as for each other aspect, mutatis mutandis.
The invention will now be exemplified. The following exemplary description and specific examples refer to the accompanying drawings, in which:
Figure 1 shows the dependence of FMDV leader protease peptidase activity as a function of alkyl glucosides detergent concentration;
Figure 2 shows the dependence of FMDV leader protease peptidase activity as a function of n-dodecyl-β-D-maltoside detergent concentration;
Figure 3 shows the dependence of FMDV leader protease peptidase activity as a function of cholate and deoxycholate detergent concentration;
Figure 4 shows the dependence of FMDV leader protease peptidase activity as a function of Big CHAP detergent concentration;
Figure 5 shows the dependence of FMDV leader protease peptidase activity as a function of Triton X- 100 detergent concentration; Figure 6 shows the dependence of FMDV leader protease peptidase activity as a function of NP-40 detergent concentration;
Figure 7 shows the dependence of FMDV leader protease peptidase activity as a function of Brij 35 and Genapol C-100 detergent concentration;
Figure 8 shows the dependence of FMDV leader protease peptidase activity as a function of Pluronic F-127 detergent concentration;
Figure 9 shows the dependence of FMDV leader protease peptidase activity as a function of Tween 20 detergent concentration;
Figure 10 shows the dependence of FMDV leader protease peptidase activity as a function of Empigen BB detergent concentration;
Figure 11 shows the dependence of FMDV leader protease peptidase activity as a function of Zwittergent detergent concentration;
Figure 12 shows the dependence of FMDV leader protease peptidase activity as a function of CHAPS and CHAPSO detergent concentration;
Figure 13 shows the dependence of FMDV leader protease peptidase activity as a function of APO-12 detergent concentration;
Figure 14 shows the dependence of FMDV leader protease peptidase activity as a function of SDS detergent concentration;
Figure 15 shows the dependence of FMDV leader protease peptidase activity as a function of Elugent detergent concentration; and
Figure 16 shows the addition of 0.5 mM deoxycholate to assay buffer prevents the loss of FMDV leader protease peptidase activity during freeze-thaw cycles. General Experimental Methods
All solvents were purchased from ROMIL Ltd. (Waterbeach, Cambridge, UK) at SpS or 'Hi-Dry' grade unless otherwise stated. General peptide synthesis reagents were obtained from Chem-Impex International Inc. (Wood Dale, IL 60191, USA). All aminoacids are of the L-configuration unless otherwise stated. All 7-amido-4- methylcoumarin (AMC) based substrates and O-succinimidyl esters were purchased from Bachem UK, St. Helens, Merseyside, U.K. All analytical HPLC were obtained on Phenomenex Jupiter C4, 5μ, 30θA, 250 x 4.6 mm, using mixtures of solvent A = 0.1% aqueous trifluoroacetic acid (TFA) and solvent B = 90% acetonitrile/10% solvent A on an automated HPLC system (Agilent Technologies, Bracknell, UK) with 215 and/or 254nm UV detection. Unless otherwise stated, a gradient of 10 - 90% B in A over 25 minutes at 1.5 ml/min. was performed for full analytical HPLC analysis. HPLC-MS analysis was performed on Agilent 1100 series LC/MSD, using automated Agilent HPLC systems (HP 1100 system; Agilent Technologies, Bracknell, UK), with a gradient of 10 - 90% B in A over 10 min. on Phenomenex Columbus C8, 5μ, 300A, 50 x 2.0 mm at 0.4 ml/min. The mass spectrometer was set to API-ES ionisation mode, positive polarity; scanning in the 100-1500 Da mass range with a gas temperature was set to 350°C. Semi-preparative HPLC purification of crude samples was performed on Phenomenex Jupiter C4 (5μ, 300 A) using a linear increasing gradient of solvent B in solvent A using the gradient indicated, at 4.0 ml/min. The eluant absorbance was monitored at 215 nm and fractions collected manually, then lyophilised.
General Solid Phase Peptide Synthesis Methods
The Substrates (1 - 7), utilizing fluorescence resonance energy transfer methodology (FRET-based substrates), were synthesised using general solid phase protocols (Atherton & Sheppard, (1989), Solid Phase Peptide Synthesis, IRL Press, Oxford, U.K), employing N-terminal 2-amino-benzoyl (Abz) as the fluorescence donor and 3-nitrotyrosine (Tyr(3-NO2)) as the fluorescence quencher (Meldal, M. and Breddam, K., (1991), Anal. Biochem., 195, 141-147). Peptide synthesis was carried out in 5 ml polypropylene plastic syringes fitted with an end-cap, a TEFLON scinter and stopper. Resin (NOVASYN TGR resin; 0.2 mmol/g; CNBiosciences, Beeston, Nottingahmshire, U.K) was added to the syringe as required. Resin was solvated in dimethylformamide (DMF) (3 -5ml) on a rotating bed (SRT1, Stuart Scientific; Fisher Scientific, Loughborough, Leicestershire, U.K) for approximately 10 min. Peptide synthesis was carried out in repetitive cycles consisting of a coupling step, a reagent wash step, an Fmoc de-protection step, then a de-protection wash step followed by the next coupling round. Between each step excess reagent and solvent were removed by application of a vacuum. Each coupling step was commenced by activating a three-mole excess of Fmoc-amino acid (with respect to total resin loading capacity) via 2-(lH-benzotriazole-l-yl)-l, 1,3,3- tetramethyluronium hexafluorophosphate (HBTU), 1-hydroxybenzotriazole (HOBt) and N-methylmorpholine (NMM). The activation mixture was pre-mixed in DMF (2- 3 ml) for 30 s and the coupling step initiated by addition to the drained resin. The syringe was capped and agitated on the rotating bed for 1 h. The excess reagents were then drained (by application of the vacuum) and the bed washed with 6 x DMF (5 ml per wash). Fmoc deprotection then commenced by continuously washing the resin bed, under gravity flow, for 10 min. with piperidine:DMF (20%:80%). The excess reagents were again drained (by application of the vacuum) and the bed washed with 10 x DMF (5 ml per wash). The resin was drained as before and ready for the next round of coupling.
Upon completion of the sequence, crude substrate cleaved with one of two cleavage cocktails for 75 min. For peptides containing Arg residues, a cocktail of 92.5% TFA:2.5% triisopropylsilane:2.5% water:2.5% ethanedithiol (40 ml/g resin) was used. For non- Arg containing peptides, a cocktail of 95% TFA:2.5% triisopropylsilane:2.5% water was used. The resin was removed by filtration and the filtrate concentrated by sparging with nitrogen gas. The crude products were precipitated by addition of 50 ml cold methyl tert-butyl ether (MTBE) and precipitates collected by centrifugation (5500 r.p.m. for 5 min). The supernatant was discarded and the process repeated. The final crude products were re-dissolved in 50:50% acetonitrile:water and analysed by RP-HPLC-MS. Crude products at >97% purity by UV analysis, were subsequently lyophilised. When required, poorer quality crude products were purified by semi-preparative HPLC and desired fractions pooled then lyophilised.
Synthesis of Abz-Lys-Leu-Cys(Bzl)-Phe-Ser-Lvs-Gln-Leu-Tyr(3-NO2 -Asp-NH2 (1)
Following the general solid phase techniques detailed earlier, NOVASYN TGR resin (0.125 g, 25 μmol) was stepwise elaborated with Fmoc-Asp(OtBu)-OH (30.9 mg, 75 μmol), Fmoc-Tyr(3-NO2)-OH (33.6 mg, 75 μmol), Fmoc-Leu-OH (26.5 mg, 75 μmol), Fmoc-Gln(Trt)-OH (45.8 mg, 75 μmol), Fmoc-Lys(Boc)-OH (35.1 mg, 75 μmol),
Figure imgf000017_0001
(28.7 mg, 75 μmol), Fmoc-Phe-OH (29.1 mg, 75 μmol), Fmoc-Cys(Bzl)-OH (32.5 mg, 75 μmol), Fmoc-Leu-OH (26.5 mg, 75 μmol), Fmoc- Lys(Boc)-OH (35.1 mg, 75 μmol) and Boc-2-Abz-OH (17.8 mg, 75 μmol), each coupling step utilising HBTU (28.4 mg, 75 μmol), HOBt (11.5 mg, 75 μmol) and NMM (6.6 μl, 75 μmol) activation.
Product (1) was cleaved and lyophilised, yield 16.2 mg (10.8 μmol, 43%), ESI-MS 692.7 [M + 2H]2+ (calc. Mw 1384.2) with Rt 6. min (>95 %).
Synthesis of Abz-Leu-Val-Gly-Arg-Ala-Gln-Tyr(3-NO2)-Asp-NH2 (2)
Following the general solid phase techniques detailed earlier, NOVASYN TGR resin (0.125 g, 25 μmol) was stepwise elaborated with Fmoc-Asp(OtBu)-OH (30.9 mg, 75 μmol), Fmoc-Tyr(3-NO2)-OH (33.6 mg, 75 μmol), Fmoc-Gln(Trt)-OH (45.8 mg, 75 μmol), Fmoc-Ala-OH (23.3 mg, 75 μmol), Fmoc-Arg(Pmc)-OH (44.8 mg, 75 μmol), Fmoc-Gly-OH (22.3 mg, 75 μmol), Fmoc-Val-OH (25.5 mg, 75 μmol), Fmoc-Leu- OH (26.5 mg, 75 μmol) and Boc-2-Abz-OH (17.8 mg, 75 μmol), each coupling step utilising HBTU (28.4 mg, 75 μmol), HOBt (11.5 mg, 75 μmol) and NMM (6.6 μl, 75 μmol) activation.
Product (2) was cleaved and lyophilised, yield 13.1 mg (12.1 μmol, 43%), ESI-MS 542.7 [M + 2H]2+ (calc. Mw 1083.16) with Rt 5.4 min (>95 %). Synthesis of Abz-Lys-Leu-Lys-Gly-Ala-Gly-Tyr(3-NO2)-Asp-NH?: (3)
Following the general solid phase techniques detailed earlier, NOVASYN TGR resin (0.125 g, 25 μmol) was stepwise elaborated with Fmoc-Asp(OtBu)-OH (30.9 mg, 75 μmol), Fmoc-Tyr(3-NO2)-OH (33.6 mg, 75 μmol), Fmoc-Gly-OH (22.3 mg, 75 μmol), Fmoc-Ala-OH (23.3 mg, 75 μmol), Fmoc-Gly-OH (22.3 mg, 75 μmol), Fmoc-Lys(Boc)-OH (35.1 mg, 75 μmol), Fmoc-Leu-OH (26.5 mg, 75 μmol), Fmoc- Lys(Boc)-OH (35.1 mg, 75 μmol) and Boc-2-Abz-OH (17.8 mg, 75 μmol), each coupling step utilising HBTU (28.4 mg, 75 μmol), HOBt (11.5 mg, 75 μmol) and NMM (6.6 μl, 75 μmol) activation.
Product (3) was cleaved, using 95% TFA:2.5% triisopropylsilane:2.5% water for 75 mins. and worked up as detailed above. The crude product was lyophilised, yield 11.9 mg (9.0 μmol, 36%), ESI-MS 507.7 [M + 2H]2+ (calc. Mw 1012.16) with Rt 4.4 min (>97 %).
Synthesis of Abz-Arg-Lys-Leu-Lys-Gιy-Ala-Gιy-Ser-Tyr(3-NO2)-Asp-NH2 (4)
Following the general solid phase techniques detailed earlier, NOVASYN TGR resin (0.125 g, 25 μmol) was stepwise elaborated with Fmoc-Asp(OtBu)-OH (30.9 mg, 75 μmol), Fmoc-Tyr(3-NO2)-OH (33.6 mg, 75 μmol), Fmoc-Serφu OH (28.8 mg, 75 μmol), HBTU (28.4 mg, 75 μmol), Fmoc-Gly-OH (22.3 mg, 75 μmol) and Fmoc- Ala-OH (23.3 mg, 75 μmol), each coupling step utilising HBTU (28.4 mg, 75 μmol), HOBt (11.5 mg, 75 μmol) and NMM (6.6 μl, 75 μmol) activation. The sixth coupling mixture consisted of Fmoc-(FmocHmb)-Gly-Opfp (61.7 mg, 75 μmol) and HOBt (11.5 mg, 75 μmol), coupling for approximately 8 h at room temperature. The seventh coupling mixture consisted of Fmoc-Lys(Boc)-OH (35.1 mg, 75 μmol), HBTU (28.4 mg, 75 μmol), HOBt (11.5 mg, 75 μmol) and NMM (6.6 μl, 75 μmol), coupling for approximately 16 h at room temperature. Synthesis then continued as normal with Fmoc-Leu-OH (26.5 mg, 75 μmol), Fmoc-Lys(Boc)-OH (35.1 mg, 75 μmol), Fmoc-Arg(Pmc)-OH (44.8 mg, 75 μmol) and Boc-2-Abz-OH (17.8 mg, 75 μmol), each coupling step utilising HBTU (28.4 mg, 75 μmol), HOBt (11.5 mg, 75 μmol) and NMM (6.6 μl, 75 μmol) activation.
Product (4) was cleaved and purified by semi-preparative HPLC using the following column elution profile: 0-1 min., 12% buffer B; 1-15 min., 12-50% buffer B; 15-17 min., 50-90% buffer B; 17-19 min., 90% buffer B; 19-21 min., 90-12% buffer B; 21- 25 min., 12% buffer B. The eluant absorbance was monitored at 230 nm and fractions were collected manually. Product containing fractions were lyophilised to give (4), yield 3.2 mg (2.5 μmol, 10%), ESI-MS 419.9 [M + 3H]3+, 629.2 [M + 2H]2+ (calc. Mw 1256.4) with Rt 4.2 min (>97%).
Synthesis of Abz-Arg-Lys-Leu-Lys-Gly- Ala-Gly-Asn-Tyr(3-NO2)-Asp-NH2 (5)
Following the general solid phase techniques detailed earlier, NOVASYN TGR resin (0.125 g, 25 μmol) was stepwise elaborated with Fmoc-Asp(OtBu)-OH (30.9 mg, 75 μmol), Fmoc-Tyr(3-NO2)-OH (33.6 mg, 75 μmol), Fmoc-Asn(Trt)-OH (44.8 mg, 75 μmol), Fmoc-Gly-OH (22.3 mg, 75 μmol), Fmoc-Ala-OH (23.3 mg, 75 μmol), each coupling step utilising HBTU (28.4 mg, 75 μmol), HOBt (11.5 mg, 75 μmol) and NMM (6.6 μl, 75 μmol) activation. The sixth coupling mixture consisted of Fmoc- (FmocHmb)-Gly-Oρfρ (61.7 mg, 75 μmol) and HOBt (11.5 mg, 75 μmol), coupling for approximately 8 h at room temperature. The seventh coupling mixture consisted of Fmoc-Lys(Boc)-OH (35.1 mg, 75 μmol), HBTU (28.4 mg, 75 μmol), HOBt (11.5 mg, 75 μmol) and NMM (6.6 μl, 75 μmol), coupling for approximately 16h at room temperature. Synthesis then continued as normal with Fmoc-Leu-OH (26.5 mg, 75 μmol), Fmoc-Lys(Boc)-OH (35.1 mg, 75 μmol), Fmoc-Arg(Pmc)-OH (44.8 mg, 75 μmol) and Boc-2-Abz-OH (17.8 mg, 75 μmol), each coupling step utilising HBTU (28.4 mg, 75 μmol), HOBt (11.5 mg, 75 μmol) and NMM (6.6 μl, 75 μmol) activation.
Product (5) was cleaved and purified by semi-preparative HPLC using the following column elution profile: 0-1 min., 12% buffer B; 1-15 min., 12-50% buffer B; 15-17 min., 50-90% buffer B; 17-19 min., 90% buffer B; 19-21 min., 90-12% buffer B; 21- 25 min., 12% buffer B. The eluant absorbance was monitored at 230 nm and fractions were collected manually. Product containing fractions were lyophilised to give (5), yield 3.2 mg (2.5 μmol, 10%), ESI-MS 428.9 [M + 3H]3+, 642.7 [M + 2H]2+ , 1284.3 [M + H]+ (calc. Mw 1283.16) with Rt 4.2 min (>95 %).
Synthesis of Abz-Ser-Val-Pro-Lys-Arg-Arg-Arg-Lys-Tyr(3-NO2)-Asp-N (6)
Following the general solid phase techniques detailed earlier, NOVASYN TGR resin (0.125 g, 25 μmol) was stepwise elaborated with Fmoc-Asp(OtBu)-OH (30.9 mg, 75 μmol), Fmoc-Tyr(3-NO2)-OH (33.6 mg, 75 μmol), Fmoc-Lys(Boc)-OH (35.1 mg, 75 μmol), Fmoc-Arg(Pmc)-OH (49.7 mg, 75 μmol), Fmoc-Arg(Pmc)-OH (49.7 mg, 75 μmol), HBTU (28.4 mg, 75 μmol), Fmoc-Arg(Pmc)-OH (49.7 mg, 75 μmol), HBTU (28.4 mg, 75 μmol), Fmoc-Lys(Boc)-OH (35.1 mg, 75 μmol), Fmoc-Pro-OH (25.3 mg, 75 μmol), Fmoc-Val-OH (25.5 mg, 75 μmol),
Figure imgf000020_0001
(28.8 mg, 75 μmol), HBTU (28.4 mg, 75 μmol) and Boc-2-Abz-OH (17.8 mg, 75 μmol), each coupling step utilising HBTU (28.4 mg, 75 μmol), HOBt (11.5 mg, 75 μmol) and NMM (6.6 μl, 75 μmol) activation.
Product (6) was cleaved (90 min) and purified by semi-preparative HPLC using the following column elution profile: 0-10 min., 10-90% buffer B; 10-12.5 min., 90% buffer B; 12.5 min., 10% buffer B. The eluant absorbance was monitored at 230 nm and fractions were collected manually. Product containing fractions were lyophilised to give, yield 4.4 mg (3.0 μmol, 12%), ESI-MS 490.0 [M + 3H]3+, 734.3 [M + 2H]2+ (calc. Mw 1466.66) with Rt 3.9 min (>97%).
Synthesis of Abz-Ala-Asn-Leu-Gly-Arg-Pro-Ala-Leu-Tyr(3-NO2)-Asρ-NH2 (7)
Following the general solid phase techniques detailed earlier, NOVASYN TGR resin (0.125 g, 25 μmol) was stepwise elaborated with Fmoc-Asp(OtBu)-OH (30.9 mg, 75 μmol), Fmoc-Tyr(3-NO2)-OH (33.6 mg, 75 μmol), Fmoc-Leu-OH (26.5 mg, 75 μmol), Fmoc-Ala-OH (23.3 mg, 75 μmol), Fmoc-Pro-OH (25.3 mg, 75 μmol), Fmoc-Arg(Pmc)-OH (49.7 mg, 75 μmol), Fmoc-Gly-OH (22.3 mg, 75 μmol), Fmoc- Leu-OH (26.5 mg, 75 μmol), Fmoc-Asn(Trt)-OH (44.8 mg, 75 μmol), Fmoc-Ala-OH (23.3 mg, 75 μmol) and Boc-2-Abz-OH (17.8 mg, 75 μmol), each coupling step utilising HBTU (28.4 mg, 75 μmol), HOBt (11.5 mg, 75 μmol) and NMM (6.6 μl, 75 μmol) activation.
Substrate (7) was cleaved and lyophilised, yield 3.2 mg (2.5 μmol, 10%), ESI-MS 626.7 [M + 2H]2+ , 1252.2 [M + H]+ (calc. Mw 1251.36) with Rt 5.4 min (>95 %).
Assays for monitoring FMDV L-peptidase peptidase activity
General materials and methods
Unless otherwise stated, all general chemicals and biochemicals were purchased from either the Sigma Chemical Company, Poole, Dorset, U.K. or from Fisher Scientific UK, Loughborough, Leicestershire, U.K. Absorbance assays were carried out in flat-bottomed 96-well plates (Spectra; Greiner Bio-One Ltd., Stonehouse, Gloucestershire, U.K) using a SPECTRAMAX PLUS384 plate reader (Molecular Devices, Crawley, U.K). Fluorescence high throughput assays were carried out in either 384-well microtitre plates (Corning COSTAR 3705 plates, Fisher Scientific) or 96-well 'U' bottomed MICROFLUOR Wl microtitre plates (Thermo Labsystems, Ashford, Middlesex, U.K). Fluorescence assays were monitored using a SPECTRAMAX Gemini fluorescence plate reader (Molecular Devices). For substrates employing either a 7-amino-4-methylcoumarin (AMC) or a 7-amino-4- trifluoromethylcoumarin (AFC) fluorophore, assays were monitored at an excitation wavelength of 365 nm and an emission wavelength of 450 nm and the fluorescence plate reader calibrated with AMC. For substrates employing a 3-amino-benzoyl (Abz) fluorophore, assays were monitored at an excitation wavelength of 310 nm and an emission wavelength of 445 nm; the fluorescence plate reader calibrated with 3- arnino-benzamide (Fluka). Unless otherwise indicated, all the peptidase substrates were purchased from Bachem UK, St. Helens, Merseyside, UK. Hydroxyethyl- piperazine ethanesulphonate (HEPES), tris-hydroxylmethylaminomethane (tris) base, bis-tris-propane (BTP) and all the biological detergents (e.g. TRITON X-100, TWEEN 20, CHAPS, β-octyl-gluopyranoside; zwittergents, etc.) were purchased from CN Biosciences UK, Beeston, Nottinghamshire, U.K. Where applicable, the commercial name and published biophysical properties of each of the detergents were used (Bhairi, S. M., (1997), Detergents: A guide to the properties and uses of detergents in biological systems, San Diego, Calbiochem-Novabiochem Corp., 675-677). Glycerol was purchased from Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, U.K. Stock solutions of substrate or inhibitor were made up to 10 mM in 100 % dimethylsulphoxide (DMSO) (Rathburns, Glasgow, U.K) and diluted as appropriately required. In all cases the DMSO concentration in the assays was maintained at less than 1% (vol./vol).
The effect of detergents on FMDV-LP activity
Recombinant wild-type FMDV-LP was obtained from Dr. Tim Skern (Institut fur Medizinische Biochemie, Abteilung fur Biochemie, Universtat Wien, Vienna, Austria). Stock solutions (10% (w/v)) of each detergent were prepared in purified water. Aliquots (5 μl) of each detergent were added to wells of row A of a 384 well microplate followed by 25 μl water to rows B to P of the same plate and a further 45 μl to row A. Row A was mixed thoroughly and 25 μl transferred to row B. The procedure was continued down the plate to produce a double dilution series down the plate from row A to row O; row P left as minus detergent controls. Separately, a stock of FMDV-LP (40 nM) was prepared in 50 mM tris-acetate, pH 8.4, 1 mM EDTA and 10 mM L-cysteine. An aliquot (12 μl) of this enzyme stock was added to each well using a multi-channel pipette, starting at row P and working up the plate. Additionally a stock solution (200 μM) of substrate (Abz-Arg-Lys-Leu-Lys-Gly-Ala- Gly-Ser-Tyr(NO2)-Asp-NH2; KM a p ~ 51 μM; Incenta Limited) was prepared in 50 mM tris-acetate, pH 8.4, 1 mM EDTA, 10 mM L-cysteine. An aliquot (12 μl) of this stock was also added to each well using a multi-channel pipette, starting at row P and working up the plate. The rate of conversion of substrate to product was derived from the slope of the increase in fluorescence monitored continuously over time at 25±1°C.
The effect of several protective surfactants is shown in the figures. The effect of detergent on the stability of FMDV-LP during freeze-thaw cycles
Samples of FMDV-LP (20 nM) were incubated in 50 mM tris-acetate, pH 8.4, 1 mM EDTA, 10 mM L-cysteine with or without 0.5 mM deoxycholate. The samples were stored in ice for 5 min. and then snap-frozen in solid carbon dioxide. The samples were stored in solid carbon dioxide for 10 min. and then subsequently thawed at 37°C (block heater info) for 10 min. Aliquots were assayed by mixing 50 μl FMDV- LP solution with 50 μl 100 μM Abz-Arg-Lys-Leu-Lys-Gly-Ala-Gly-Ser-Tyr(NO2)- Asp-NH2 in 50 mM tris-acetate, pH 8.4, 1 mM EDTA, 10 mM L-cysteine with or without 0.5 mM deoxycholate.
The effect of detergent on the stability of FMDV-LP
Samples of FMDV-LP (20 nM) were incubated in 50 mM tris-acetate, pH 8.4, 1 mM EDTA, 10 mM L-cysteine with or without 0.5 mM deoxycholate. The samples were incubated at room temperature (~ 20°C) and 37°C. Aliquots were assayed by mixing 50 μl FMDV-LP solution with 50 μl 100 μM Abz-Arg-Lys-Leu-Lys-Gly-Ala-Gly- Ser-Tyr(NO2)-Asp-NH2 in 50 mM tris-acetate, pH 8.4, 1 mM EDTA, 10 mM L- cysteine with or without 0.5 mM deoxycholate.
The results are shown in Figure 16.
Trademarks
Zwittergent® is a registered trademark of Calbiochem-Novabiochem Corporation Triton® is a registered trademark of Rohm and Haas Company BRIJ® is a registered trademark of ICI Americas Incorporated EMPIGEN® is a registered trademark of Albright & Wilson Limited Genapol® is a registered trademark of Hoescht AG PLURONIC® is a registered trademark of BASF Corporation ELUGENT™ is a registered trademark of Calbiochem-Novabiochem Corporation

Claims

1. A method of assaying foot-and-mouth disease virus (FMDV) leader peptidase activity, the method comprising assaying the peptidase activity in the presence of a protective surfactant.
2. A method as claimed in claim 1, wherein the protective surfactant is an alkyl glycoside of the general formula
R-O-(CH2)x-CH3
where R represents a mono- or di-saccharide residue and x is a number from 4 to 15.
3. A method as claimed in claim 1, wherein the protective surfactant is an alkyl thioglycoside of the general formula:
R-S-(CH2)X-CH3
where R represents a mono- or di-saccharide residue and x is a number from 4 to 15.
4. A method as claimed in claim 1, wherein the protective surfactant is a bile acid of the general formula:
Figure imgf000024_0001
where R represents a group capable of forming an anion, which may be paired with an appropriate cation.
5. A method as claimed in claim 1, wherein the protective surfactant is a glucamide of the general formula:
Figure imgf000025_0001
where n is a number from 5 to 15.
6. A method as claimed in claim 1, wherein the protective surfactant is a glucamide of the general formula:
Figure imgf000025_0002
7. A method as claimed in claim 1, wherein the protective surfactant is a polyoxyethylenes of the general formula:
Figure imgf000025_0003
where n is a number from 5 to 15.
8. A method as claimed in claim 1, wherein the protective surfactant is a polyoxyethylene of the general formula
Figure imgf000026_0001
where n is a number from 5 to 15.
9. A method as claimed in claim 1, wherein the protective surfactant is a polyoxyethylene of the general formula
CH3(CH2)y-O(CH2CH2O)xH
where x is a number from 3 to 30 and y is a number from 5 to 15.
10. A method as claimed in claim 1, wherein the protective surfactant is a polyoxyethylene of the general formula
HO(CH2CH2O)x-(CH(CH3)-CH2O)y-(CH2CH2O)z-H
where x is a number from 50 to 150 and y is a number from 30 to 100 and z is a number from 50 to 150.
11. A method as claimed in claim 1, wherein the protective surfactant is a polyoxyethylene of the general formula
Figure imgf000026_0002
where w is a number from 10 to 30, x is a number from 10 to 30, y is a number from 10 to 30, z is a number from 10 to 30 and R is an acyl group containing from 2-30 carbon atoms. i
12. A method as claimed in claim 1, wherein the protective surfactant is n-dodecyl- N,N-dimethylgrycine.
13. A method as claimed in claim 1, wherein the protective surfactant is a of the general formula:
Figure imgf000027_0001
where x is a number from 5 to 25.
14. A method as claimed in claim 1, wherein the protective surfactant is a zwitterionic detergent of the general formula:
Figure imgf000027_0002
where R is H or OH.
15. A method as claimed in claim 1, wherein the protective surfactant is zwitterionic surfactant selected from amidosulphobetaine (ASB-14 and ASB-16); DDMAU (N-dodecyl-N,N-(dimethylammomo)undecanoate); and DDMAB (N- dodecyl-N,N-(dimethylammonio)butyrate).
16. A method as claimed in claim 1, wherein the protective surfactant is a dimethyl(C5-C15)alkylρhosρhine oxide, such as APO-8; APO-9; APO-10
(dimethyldecylphosphine oxide); APO-11 and APO-12 (dimethyldodecylphosphine oxide).
17. A method as claimed in claim 1, wherein the protective surfactant is sodium n- dodecyl sulphate (SDS).
18. A method as claimed in claim 1, wherein the protective surfactant is a mixture of alkyl glucosides such as Elugent.
19. A method as claimed in claim 1, wherein the protective surfactant is TOPPS (3-([4-tert-octyl]phenoxy)-l-propane sulphonic acid sodium salt)
20. A method as claimed in claim 1, wherein the protective surfactant is a non- detergent sulphobetaine.
21. A method as claimed in any one of claims 1 to 20, wherein the peptidase activity is assayed by determining the rate of conversion of substrate to product and the substrate is labelled.
22. A method as claimed in claim 21, wherein the substrate is labelled with a FRET pair.
23. A method as claimed in claim 22, wherein the substrate is Abz-Arg-Lys-Leu- Lys-Gly-Ala-Gly-Ser-Tyr(NO2)-Asp-NH2.
24. A method as claimed in any one of claims 1 to 23, wherein the peptidase activity is assayed in the presence of a candidate modulator of peptidase activity.
25. A preparation of FMDV leader peptidase including a protective surfactant.
26. A preparation as claimed in claim 25, wherein the protective surfactant is as defined in any one of claims 2 to 20.
PCT/GB2003/000164 2002-01-17 2003-01-16 Fmdv leader peptidase assay WO2003062461A1 (en)

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EP1878801A1 (en) * 2005-05-06 2008-01-16 Arkray, Inc. Protein cleavage method and use thereof
EP1878801A4 (en) * 2005-05-06 2008-06-11 Arkray Inc Protein cleavage method and use thereof
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CN111944770B (en) * 2020-08-27 2022-04-15 中国农业科学院兰州兽医研究所 Foot-and-mouth disease virus attenuated mutant strain and preparation method and application thereof

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