MXPA99006000A - Improved substrates for human cytomegalovirus protease - Google Patents
Improved substrates for human cytomegalovirus proteaseInfo
- Publication number
- MXPA99006000A MXPA99006000A MXPA/A/1999/006000A MX9906000A MXPA99006000A MX PA99006000 A MXPA99006000 A MX PA99006000A MX 9906000 A MX9906000 A MX 9906000A MX PA99006000 A MXPA99006000 A MX PA99006000A
- Authority
- MX
- Mexico
- Prior art keywords
- substrate
- tbg
- ala
- asn
- protease
- Prior art date
Links
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Abstract
La proteasa del citomegalovirus humano (HCMV) es una enzima de procesamiento lento in vitro. En la presente se describen los sustratos peptídicos fluorogénicos y radiométricos mejorados para la proteasa de CMV. Los sustratos mejorados tienen un Tbg-Tbg-Asn(NMe2) que reemplaza la secuencia Val-Val- Asn, correspondiente a los residuos P4-P2 del sitio de maduración de la enzima. La incorporación de esta nueva secuencia en una variedad de sustratos ha proporcionado sustratos con parámetros cinéticos mejorados en comparación a los homólogos que contienen la secuencia naturalúnicamente. Por ejemplo, el sustrato 2-aminobenzoil-Tbg- Tbg-Asn(NMe2)-Ala/Ser-Ser-Arg-L eu-Tyr(3-NO2)Arg-OH mostróun valor kcat/Km de 15940 M-1s-1, por ejemplo arriba de 60 veces mayor que aquel del sustrato no optimizado, equivalente, en condiciones idénticas. Los nuevos sustratos encuentran uso en ensayos que pueden evaluar y caracterizar los inhibidores de la_proteasa de HCMV que tienen potencia nanomolar baja.
Description
"IMPROVED SUBSTRATES FOR PROTEASA DE CITOMEGALOVIRÜS HUMANA" FIELD OF THE INVENTION
This invention relates to substrates for herpesvirus proteases, to methods for determining the activity of herpesvirus proteases and to methods for identifying inhibitors of herpervirus proteases.
BACKGROUND OF THE INVENTION
Herpesviruses inflict a wide range of diseases against humans and animals. For example, herpes simplex viruses types 1 and 2 (HSV-1 and HSV-2) are responsible for cold sores and genital lesions, respectively; Varicella zoster virus (VZV) causes chickenpox and herpes; the Epstein-Barr virus (EBV) causes infectious mononucleosis; and the human cytomegalovirus (HCMV, a ß-herpervirus) is a serious pathogen in immunocompromised individuals, including AIDS patients, neonates and patients with organ tranntation (Mocarski, E.S. (1996) in Virology (Fields, B.N.,
REF .: 30647 Knipe, D.M., Howley, P.M., Eds.), Pp. 2447-2492, Lippincott-Raven Publishers, Philadelphia; and Britt, .J., Alford, C.A. (1996) in Virology (Fields, B.N., Knipe, D.M., Howley, P.M., Eds.), Pp. 2493-2523, Lippincott-Raven Publishers, Philadelphia). The most widely used anti-herpes agents to date are acyclovir and ganciclovir (purine nucleoside analogs and pyrimidine) and foscarnet. These agents have only found limited success in the treatment of herpesvirus infections, and safer and more effective treatment agents are required. As a member of the Herpesvirus family, HCMV codes for a unique serine protease, which is involved in the assembly of the capsid and is essential for the production of infectious virions (Preston, VG, Coates, JAV, Rixon, FJ (1983) J. Virol 45, 1056-1064; Gao, M., Matusick-Kumar, L., Hurlburt,., DiTusa, SF, Newcomb, W., Brown, JC, McCann, PJ, III, Deckman , I., Colonno, RJ (1994) J. Vi, 68, 3702-3712, Matusick-Kumar, L., McCann, PJ, III, Robertson, BJ, Newcomb, .W., Brown, JC, Gao, M. 1995 J. Vi rol., 69, 7113-7121; Gibson, W., Welch, AR, Hall, MRT (1995) Perspect, Drug Di verse, Des 2, 413-426; and Liu, F., Roiz an , B. (1991) J. Vi rol., 65, 5149-5156). This enzyme is responsible for the processing of the assembly protein whose function is analogous to that of the "scaffolding" protein of the bacteriophages (Casjens, S., King, J. (1975) Annu., Rev. Bi ochem. 44, 555 -611). The deficiency in processing the assembly protein results in the accumulation of only non-infectious, aberrant capsids (Preston, V.G. et al., See below). This protease therefore represents an attractive target for the development of new antiviral agents. In order to identify inhibitors of herpesvirus proteases, tests are required that allow the measurement of protease activity. The tests by initial tests were based on the electrophoretic separation of the cleavage products (Gibson, et al., See below and Liu, F. et al., See below). However, these tests were very slow and problematic. Subsequently, faster and higher performance tests were developed using fluorogenic substrates for the HCMV protease (Holskin, BP, Bukhtiyarova, M., Dunn, BM, Baur, P., de Chastonay, J., Pennington, MW (1995) Anal Bi ochem 227, 148-155; Pinko, C, Margosiak, SA, Vanderpool, D., Gutowski, JC, Condom, B., Kan, C.-C. (1995 J. Bi ol. Chem. 270, 23634-23640; Handa, BK, Keech, E., Conway, EA, Broadhurst, A., Ritchie, A. (1995) An ti vir. Chem. Chemother, 6, 255-261; and Toth, MV, Wittner, AJ, Holwerda, BC, US Patent No. 5,506,115 issued April 9, 1996.) These fluorogenic substrates are based on the maturation cleavage site of the enzyme (the "M site") and exploit the spectral overlapping properties of fluorescent donor / acceptor pairs such as EDANS / DABCYL and 2-aminobenzoic acid / 3-nitrotyrosine (Matayoshi, ED, Wang, GT, Krafft, G., Erickson, J. (1990) Sci en 247, 954-958; Meldal, M ., Breddam, K. (1991) Anal. Bi och em. 195, 141-147). The spacing between the pair in these HCMV protease substrates varies from 10 to 11 amino acids and the specificity constant, kcat / Km, is in the range of 800 to 3000 M "1s" 1 (Table I).
TABLE I
PREVIOUS REPORTS OF FLUOROGENIC SUBSTRATES OF
PROTEASA DE HCMV Substrate • Kcat? "(ΜM) cat / M (s-1) (M_1s_1)
DABCYL-RGWNA-SSRLA-EDANS to nd nd 796
DABCYL-RRWNA-S-Aba- Nd 3.0 Nd
RLD (EDANS) -NH2 b - ABz-GWNA-SSRLAY (3-N02) G c 0.37 134 2750
Y (3 -N02) GWNA- S SRLA-AB z -K c Nd 3167
to. _ Holskin, B.P., Bukhtiyarova, M., Dunn, B.M., Baur, P., de Chastonay, J., Pennington, M.W. (1995) Anal. Biochem. 227, 148-155. b. Handa, B.K., Keech, E., Conway, E.A., Broadhurst, A., Ritchie, A. (1995) Antivir. Chem. Chemother. 6, 255-261. c. Pinko, C., Margosiak, S.A., Vanderpool, D., Gutowski, J.C., Condom, B., Kan, C.-C. (1995) J. Biol. Chem. 270, 23634-23640.
These numbers reflect the extensive requirement of herpesvirus proteases for a long peptide chain and their relatively low activity compared to other viral proteases (Gibson, W. et al., See below). As described herein, a new class of substrates, modeled on the site of enzyme maturation, has been developed. The kinetic properties of the new substrates of this class are suitable for the elaboration of, for example, an assay or fluorometric, chromogenic or radiometric test to select and mechanize studies of HCMV protease inhibitors. If rapid progress is to be made in the rational design of peptide-based inhibitors, a more active substrate than those found in previous reports is required. The benefits that accumulate for a more active substrate are a significant reduction of the enzyme concentration in the assay and the allowance of an unambiguous determination of the potency of the inhibitor. The improved activity requirement has now been met by the new substrates described herein. These new substrates are specifically useful for high performance liquid chromatography (HPLC), radiometric, chromogenic, and fluorometric based assays. The latter type of assay usually offers significant advantages over other procedures, such as increased yield, increased sensitivity and continuous verification of the hydrolytic process. More recently, the increasing interest in the design of new fluorogenic substrates is exemplified by their numerous application in a variety of enzymatic protocols (Matayoshi, ED et al., See below; Wang, GT, Chung, CC, Holzman, TF, Krafft, GA (1993) Anal. Bihochem 210, 351-359; Pennington, NW, Zaydenberg, I., Byrnes, ME, Chastonay, J., Malcolm, BA, Swietnicki, W., Farmerie, WG, Scarborough, PE , Dunn, BM (1993) in Peptides 1992; Proceedings of the 22nd European Peptide Symposium (Schneider, CH, Eberle, AN, Eds), pp. 936-937, Escom, Leiden, The Netherlands, Pennington, MW, Thornberry, NA ( 1994) Pepti of Res. 1, 72-76, Knight, GC (1995) Meth. Enzymol, 248, 18-34 (and references herein), and Jean, F., Basak, A., DiMaio, J. , Seidah, NG, Lazure, C. (1995), Bi ochem J. 307, 689-695). European Patent 0,493,770 describes the peptide PhCH2CH2CO-N-Me-Tbg-Tbg-Asp (NEt2) -Asp (diMe) -Leu-OH. This peptide is directed towards the enzyme ribonucleotide reductase of the herpesvirus and does not fall under the present invention.
BRIEF DESCRIPTION OF THE INVENTION
One aspect of this invention involves a suitable substrate for cleavage or cleavage for a herpesvirus protease, represented by formula I:
X-Tbg-Tbg-Y-Ala-Z (I) [SEQ. ID. No. 1]
wherein X is a fluorescent donor moiety, a fluorescent acceptor moiety, an affinity tag, a detectable tag, or an N-terminal clustering group; or X is a sequence of amino acids, or a derivative thereof, sufficient for recognition of the substrate and to which a fluorescent donor moiety, a fluorescent acceptor moiety, an affinity tag, a detectable tag or a clustering group is optionally coupled. N-terminal; Y is a divalent radical NHCH [CH2C (O) N (R1) (R2)] C (0) wherein R1 and R2 are independently selected from hydrogen, alkyl of 1 to 6 carbon atoms, lower cycloalkyl or (lower cycloalkyl) - (alkyl of 1 to 6 carbon atoms), or R1 and R2 together with the hydrogen atom to which they are attached form a pyrrolidino, piperidino or morpholino; and Z is an amino acid sequence or derivative thereof, sufficient for recognition of the substrate and to which is optionally coupled a fluorescent donor moiety, a fluorescent acceptor moiety, an affinity tag or a detectable tag; or Z is an indo compound or aromatic phenoxy type whose fluorescence or absorbance characteristics change after acylation with an amino acid; or Z is amino, alkylamino of 1 to 6 carbon atoms or lower alkoxy; with the proviso that (1) when one of X and Z comprises a fluorescent donor then the other of X and Z comprises a fluorescent acceptor radical; and (2) that when one of X and Z comprises an affinity tag then the other of X and Z comprises a detectable tag; wherein the protease breaks the substrate at the corresponding amide or ester linkage between Ala and Z. According to yet another aspect of the present invention, methods are provided for measuring the activity of a herpesvirus protease using the aforementioned substrate.
According to yet another aspect of the present invention, methods for identifying inhibitors of a herpervirus protease using the aforementioned substrate are provided.
DESCRIPTION OF THE FIGURE
Figure 1 illustrates the inhibitory effect of a peptide inhibitor on the HCMV protease, at various concentrations of an improved substrate of this application.
DETAILED DESCRIPTION OF THE INVENTION
In general, the abbreviations used herein to designate amino acids and protecting groups are "based on the recommendations of the IUPAC-IUB Biochemical Nomenclature Commission, see European Journal of Biochemistry 138, 9 (1984). example, Ala, Arg, Val, Lie, Ser, Tbg, Thr, Tyr, Asn and Leu represent the residues of L-alanine, L-arginine, L-valine, L-isoleucine, L-serine, L-tert-butylglycine , L-threonine, L-tyrosine, L-asparagine and L-leucine, respectively The term "fluorescent donor moiety", as used herein, means a fluorescence-emitting moiety which can be encoded and coupled to the sequence Examples of such radicals are those derived from 2-aminobenzoyl (and halogenated derivatives thereof), 5- ({(2-aminoethyl) amino} -naphthalene-1-sulfonyl (EDANS), 5- ( dimethylamino) naphthalene-1-sulfonyl
(DANSYL), 7-methoxycoumarin-4-acetyl, nicotinic acid (and derivatives thereof) and tryptophan. The term "fluorescent acceptor radical", as used herein, means an aromatic quenching radical which absorbs the fluorescent energy of the fluorescence donor moiety and reduces fluorescence emission when the fluorescence donor moiety is covalently linked in close proximity. to the radical acceptor. Examples of such radicals include 3-nitrotyrosine, 4-nitrophenylalanine, 2,4-dinitrophenylalanine, DANSYL, 4-. { . { 4- (dimethylamino) phenyl} azo } benzoyl (DABCYL) or 4- (dimethylamino) azobenzene-4'-sulfonyl (DABSYL). The term "affinity tag", as used herein, means a ligand whose strong affinity for a receptor can be used to extract from a solution the entity to which the ligand is covalently linked. Examples of such ligands include biotin or a derivative thereof, a histidine polypeptide, an amylose sugar moiety or a defined epitope recognized by a specific antibody. The term "detectable label", as used herein, means an atom or a radical whose physical properties or interactions with other molecules allow the sensitive detection of its presence by radioactive or chromogenic measurements. The term "grouping group" ', as used herein, means a group that replaces a hydrogen on the N-terminal nitrogen of an amino acid or peptide. The group has the effect of preventing additional chemical reactions from occurring at that site until the stacking group is ultimately eliminated. Examples of grouping groups include acetyl and succinimidyl. The term "aromatic amino radical", as used herein, means a radical derived from an aromatic amine by the removal of a hydrogen from the amino group; the aromatic amine having fluorescence or absorbance properties that change after acylation of the amino group. Examples of aromatic amines include 7-amino-4-methylcoumarin, 6-amino-1-naphthalenesulfonamine, rhodamine 110 (Aldrich Chemical Co., Milwaukee, Wl, USA), 2-naphthylamine, 4-methoxy-2-naphthylamine and nitroaniline. . The term "phenoxy radical", as used herein, means a radical derived from a phenolic compound by the removal of a hydrogen from the hydroxyl, the phenolic compound having fluorescence or absorbance properties that change after acylation of the hydroxyl. Examples of such phenolic compounds include 4-nitrophenol and the like. The term "lower alkyl" as used herein, either alone or in combination with a radical, means straight chain alkyl radicals containing one to six carbon atoms (alkyl of 1 to 6 carbon atoms) and alkyl radicals branched chain containing three to four carbon atoms and include methyl, ethyl, propyl, butyl, hexyl, 1-methylethyl, 1-methylpropyl, 2-methylpropyl and i, 1-dimethylethyl.
The term "lower cycloalkyl" as used herein, either alone or in combination with a radical, means the saturated, cyclic hydrocarbon radical containing from 3 to 6 carbon atoms and includes cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. The term "lower alkoxy" as used herein, means a straight chain alkoxy radical containing one to six carbon atoms and branched chain alkoxy radicals containing three to four carbon atoms, and include methoxy, ethoxy, propoxy , butoxy, hexoxy, 1-methylethoxy and 1,1-dimethylethoxy. The last radical is commonly known as tert-butyloxy. With additional reference to the substrates and assays of this invention, the HCMV protease is an endoproteolytic enzyme that breaks down its precursor protein at different multiple sites
(Maturation, Liberation, and Interi orization) having the general consensus V- (N / E / K) -R-I (S / A) (Gibson, W. et al., See below). The efficient hydrolysis by the HCMV protease of the peptide substrates corresponding to the natural cleavage sites generally requires the minimum amino acid sequence P4 to P'4. In addition, the enzyme breaks the peptides from the maturation site up to 30 times more efficiently than those corresponding to the release site. (The maturation sequence was therefore chosen as the starting point for the synthesis of the substrates and the development of peptide-based inhibitors). The present initial fluorogenic substrate having the structure of Formula 1:
ABz-Val-Val-Asn-Ala-Ser-Ser-Arg-Leu-Tyr (3-N02) Arg-OH (1) [SEQ. ID. NO .: 2]
used the anthranilamide / nitrotyrosine system. This choice considered the excellent properties of spectral overlap of this donor / acceptor pair and the ease of synthesis by solid-phase chemistry. { Mendal, M. et al., See below and Gron, H., Mendal, M. Breddam. K. (1992), Bi ochemi s try 31, 6011-6018} . The substrate of formula 1 encompassed the P4-P '4 sequence of the maturation site in which the P' 2 cysteine residue was replaced by serine to prevent oxidation problems. The anthranilamide portion conveniently encapsulated the N-terminus and while the 3-nitrotyrosine was placed eight residues away, preceding the C-terminal arginine residue which was incorporated to increase the solubility. The relatively short distance between the donor / acceptor pair and the nature of the amino acid sequence allowed efficient internal fluorescence quenching. Of course the Fo / Fa ratio was "4% after excitation at 312 nm and emission at 415 nm (Fo and F" are the measured fluorescence for the substrate and the N-terminal hydrolysis product respectively at equal concentrations) . In addition, no substantial intermolecular quenching of the fluorescence by excess substrate interfered with the signal below 15 μM (internal filter effect), consistent with the first reports for this class of substrates (Mendal, M. et al., See below). ). Above this concentration a significant internal filter effect became apparent. The kcat and Km kinetic parameters for the substrate of formula 1 in the presence of 0.5 M Na2SO4 using the 50 nM HCMV protease were 0.20 s-1 and 760 μM respectively as measured by HPLC (Table II).
TABLE II
KINETIC PARAMETERS OF PROTEASE SUBSTRATES
HCMV Substrate k cat (S) KM (μM) Kcat / KM (M ^ s-1)
• i a, c 0.20 760 260 2a '0.051 3.2 15940 3a' d 0.035 13.2 2650 4b- G 0.086 422 205 5b, c 0.045 19 2370 a Using 50 nM HCMV protease (total monomer concentration). b Using HCMV protease 200-500 nM. c Determined by HPLC. d Determined by fluorometry. The resulting specificity constant cat / m of 260 M "1s" 1 compares favorably with other substrates of the maturation site (Gibson, W. et al., See below). However, the limited solubility of the substrate of formula 1 and its high value of Km limited the accuracy of the individual parameters and did not allow determinations of the kinetic parameters by fluorescence due to the internal filter effect. However, below this concentration the substrate can conveniently periodically verify the protease activity of HCMV. The substrate of formula 1 was used to implement a 100 nM HCMV protease assay for routine selection of inhibitors. However, the rapid progress in studies of structure-activity relationship (SAR) eventually required a reduction of the enzyme concentration in order to differentiate between the potent compounds. Of course, an IC5o value can not be less than half the enzyme concentration in the assay. By reducing the enzyme, therefore, the precise determination of the potency of the active inhibitors is allowed. It was found that the direct introduction of Tbg-Tbg-Asn (Me) 2 instead of the natural Val-Val-Asn P4-P2 sequence within the substrate of formula 1, gave a more efficiently cleaved fluorogenic substrate having the structure of Formula 2:
ABz-Tbg-Tbg-As (Me) 2-Ala-Ser-Ser-Arg-Leu-Tyr (3-N02) -Arg-OH (2) [SEQ. ID. NO: 3]
The fluorescent properties of the substrate of formula 2 were similar to those of the substrate of formula 1 but their kinetic characteristics were significantly improved. The kcat and Km kinetic parameters for the substrate of formula 2 under the standard conditions were 0.051 s-1 and 3.2 μM respectively, as measured by fluorometry (Table II). The resulting specificity constant kcat / Km of 15940 M-1s-1 was 60 times higher than for the non-optimized substrate. The large reduction in Km was responsible for this substantial increase in activity, and reflected the anticipated improvement in the link. The inhibitory activity of the N-terminal product ABz-Tbg-Tbg-Asn (Me) 2-Ala-OH was determined to establish whether it could interfere with the course of the reaction. An IC 50 value above 75 μM clearly indicated that its influence could not be detected significantly in the range of substrate concentration used. These improved kinetic parameters gave the substrate of formula 2 a number of significant advantages over other existing fluorogenic substrates. Firstly, the substrate easily allowed the development of an assay using only the 5 nM HCMV protease, thus allowing unequivocally characterized nanomolar low potency inhibitors to be characterized. Secondly, the substrate concentrations significantly above the value of Km but below the limit imposed by the internal filter effect were accessible for the periodic verification of the continuous fluorescence, thus allowing a variety of analysis to be carried out rigorously. kinetics, simply and quickly. To date, only tedious and time-consuming HPLC protocols could be used for certain applications, since none of the well-characterized, available fluorogenic protease substrates of HCMV showed fluorescent properties compatible with measurements above Km. { Margosiak, S.A., Vanderpool, D.L., Sisson, W., Pinko, C., Kan, C.-C.
(1996) Bi ochemi s try 35, 5300-5307). Such measurements are essential for the precise determination of the individual values of kcat and Km and for the analysis of the modes of inhibition. For example, the mode of inhibition of the peptide-based ketoamide inhibitor, of the protease N0 of HCMV, DATbg-Tbg-Asn (Me2) -Ala-CONHCH2Ph
[I KNOW THAT. ID. NO: 4] was shown to be competitive by Dixon plot analysis of the initial rate of hydrolysis at different concentrations of substrate 2 and inhibitor (Figure 1). The convenient use of fluorescence in this experiment was possible since a range of substrate concentrations of 0.4 to 2.5 km was allowed. Substrate 1 could not be used in this application since its Km value is well above the internal filter limit. The aforementioned ketoamide inhibitor was prepared by the procedure of Example 9. The most important factors limiting the use of these internally quenched fluorogenic substrates are the presence of the internal filter effect around 15-20 μM and the background of significant fluorescence. at low concentrations. In an attempt to further improve the present arsenal of fluorogenic substrates, it was decided to introduce the fluorescent group 7-amino-4-methylcoumarin (AMC) into the cleavable link. { Zimmerman, M., Yurewicz, E., Patel, G. (1976) Anal. Bi ochem. 70, 258-262). AMC has been successfully used in a variety of fluorescent cysteine and serine protease substrates (Proteolytic Enzymes: Serine and Cysteine Peptidases (1994) in Meth. Enzymol 244, (Barrett, AJ, Ed.), Pp. 1- 765.}. Its main advantage relies on a high signal-to-noise ratio as fluorescence appears only after excision, and does not require any other accessory group.Addition of this fluorophore directly on the alanine residue Pi resulted the replacement of P 'residues by the AMC portion Although these residues are essential in natural peptide substrates, it was reasoned that the optimized sequence Tbg-Tbg-Asn (Me) 2 P4-P2 can largely compensate for its replacement by the AMC group The substrate that has the formula 3:
N-Ac-Tbg-Tbg-Asn (Me) 2-Ala-AMC (3) [SEQ. ID. NO: 5]
was synthesized as a representative member of a new family of fluorogenic, short HCMV protease substrates. The substrate of formula 3 showed excellent kinetic properties with kcat and Km values under the test conditions of 0.035 s "1 and 13.2 μM respectively, as measured by fluorometry (Table II). The resulting specificity constant, Kcat / Km, 2650 M-1s_1 was 10 times higher than for the substrate of formula 1 despite lacking four P 'residues and only 6 times smaller than the substrate of formula 2.
The 'substrate of formula 3 represents the first demonstration that the HCMV protease (and probably all other herpesvirus proteases), do not absolutely require the presence of P' amino acids for efficient cleavage. The intense fluorescence signal generated by the release of the AMC group combined to a negligible antecedent, confer to the substrate of formula 3 the advantages of easy detection of cleavage on instruments of low sensitivity and accessibility to a wide range of concentrations within the limitations of solubility (Knight, GC et al., see below). More specifically, according to the present invention, the improved substrates of the formula I and the assays for herpesvirus protease are described using the improved substrates. The assays can be used to measure the activity of the CMV protease, to bring the herpesvirus protease in contact with the improved substrate and periodically verify the cleavage or cleavage of the substrate. A preferred class of substrates is represented by formula I wherein Z is an aromatic amino radical or a phenoxy radical derived from 7-amino-4-methylcoumarin, 6-amino-1-naphthalenesulfonamide, rhodamine 110, 2-naphthylamine, 4 -methoxy-2-naphthylamine, 4-nitroaniline or 4-nitrophenol. Another preferred class of substrates includes substrates of formula I wherein the substrate is covalently linked to a fluorescent donor moiety and a fluorescent acceptor moiety, the donor and acceptor moieties being separated by the alanine residue and 7 to 12 additional amino acid residues. . Another preferred group of substrates is represented by formula I wherein the radical donor / acceptor pair is selected from the following pairs: EDANS / DABCYL, tryptophan / 2,4-dinitrophenyl, tryptophan / DANSYL, 7-methoxycoumarin / 2, 4 -dinitrophenyl, 2-aminobenzoyl / 2,4-dinitrophenyl or 2-aminobenzoyl / 3-nor trotyros ina. Other useful substrates of this invention. are those linked to a detectable marker and an affinity tag or label. Practical and useful detectable labels are radioactive labels or markers such as 125I, or β-galactosidase, para-nitrophenol or para-ni troaniline.
Useful and useful affinity labels are those derived from biotin (or derivatives thereof), polyhistidine, a portion of amylose sugar or a defined epitope recognizable by a specific antibody. Such labels and affinity tags are coupled to the substrate by well-known methods. A method for measuring a herpes protease, as well as a method for identifying an inhibitor of a herpes virus protease, are described in more detail and are exemplified hereinafter. The following examples further illustrate this invention. Regarding the examples please note the following: All the natural amino acid derivatives and the N-α-Fmoc-NG-pMc-L-Arginine-Wang resin were purchased from Bachem (Torrance, California, USA). 3-Nitro-L-tyrosine was purchased from Sigma Chemical Co. , Saint Louis, MO, USA, and 2-aminobenzoic acid
(ABz) and 7-amino-4-methylcoumarin (AMC) were purchased from Aldrich Chemical Co. , Milwaukee, Wl,
USA and the compounds were protected by standard methods. Peptide substrate 1 was prepared on an Advanced Chemtech peptide synthesizer model ACT396 / 5000 MPS. Substrate 2 was prepared using a Coupler® 250C agitator-type peptide synthesizer from Vega Biotechnologies (Dupont Co., Wilmington, DE., USA). The 1 H NMR spectra were recorded on a Bruker 400 MHz spectrometer, the chemical shifts (d) are reported in parts per million. The analytical HPLCs were performed on a Waters instrument equipped with a Vydac C18 reversed phase column, 0.46 x 25 cm, 5 μm, 300 A (gradient: 5 to 65% acetonitrile / water containing 0.06% trifluoroacetic acid at 25 ° C). minutes at a flow rate of 1.5 ml / minute). The amino acid analysis was performed using the PICO-TAG® method using a Waters HPLC system. For enzymatic kinetics, HPLC determinations were performed using the Perkin-Elmer Xpress 3X3CR C8 reverse phase cartridge columns. The fluorescence measurements were made in quartz cuvettes in a Perkin-Elmer LS50B luminescence spectrophotometer. Abbreviations or symbols used in the examples, or throughout the present specification include Aba, 2-aminobutyric acid; ABz, 2-aminobec acid or anthranilic acid; Ac, acetyl; AMC, 7-amino-4-methylcoumarin; As (Me) 2, 2 (S) -amino-4- (dimethylamino) -4-oxobutanoic acid; Boc, tert-butyloxycarbonyl; DABCYL, acid 4-. { . { 4- (dimethylamino) phenyl} azo } bec; DANSYL, 5- (dimethylamino) naphthalene-1-sulfonyl; DATbg, desamino-tert-butylglycine (3, 3-dimethylbutylbutanoic acid); DCC N, N '-dicyclohexylcarbodiimide; DIC, 2-dimethylaminoisopropyl chloride hydrochloride; DIPEA, diisopropylethylamine; DMF, N, N, -dimethylformamide; DMSO, dimethylsulfoxide; DTT, dithiothreitol; EDANS, acid 5-. { (2-aminoethyl) -amino} Naphthalene-1-sulfonic acid; Et20, diethyl ether; EtOAc, ethyl acetate; Fmoc, 9-fluorenylmethyloxycarbonyl; HCMV, human cytomegalovirus; HOBT, 1-hydroxyberiazole hydrate; IPTG, isopropyl-β-D-thiogalactopyranoside; MeOH, methanol; MONTH, 4-morpholinoethane sulfonic acid; NMP, N-methylpyrrolidone; PCR, polymerase chain reaction; Ph, phenyl; Pmc, 2,2,5,7,8-pentamethylchroman-6-sulfonyl; PMSF, phenylmethylsulfonyl fluoride; QSAR, quantitative relation of structural activity; Tbg, tert-butylglycine; tBu, tert-butyl; TFA, trifluoroacetic acid; Trt, triphenylmethyl; TBTU, O- (beriazol-1-yl) -1, 1,3,3-tetramethyluronium tetrafluoroborate; TCEP, tris (2-carboxyethyl) phosphine hydrochloride; TRIS, tris (hydroxymethyl) aminomethane; Tyr (3-N02), 3-nitrotyrosine. The substrate positions are denoted
Pi, P2, Pi ^ P1 i, Z i 3 'where the cleavage or break occurs in the arrow (Schechter, J., Berger, A. (1970) Phil os, Trans. R. Soc. London, Ser. B, 251, 249-264).
Example 1
Preparation of recombinant wild-type HCMV protease
The HCMV protease gene amplified by -PCR, from genomic DNA (UL80 gene from
HCMV strain AD169, amino acids 1-256) was cloned into the Ndel / BamHI restriction sites of the plasmid vector pET-17b and expressed in Escheri chi a Col i
BL21 (DE3) pLYSs. After expression by induction with IPTG, the cells were harvested and resuspended in lysis buffer (50 mM Tris / HCl pH 8.0, 1 M EDTA, 1 mM DTT, 25 mM sodium chloride, and 1 mM PMSF) and sonicated for complete lysis and DNA breakage. The protease inclusion bodies were harvested by centrifugation.
The harvested material was washed twice in lysis buffer containing 0.1% octylphenyl ether of nonaethylene glycol (NP-40, Sigma Chemical Co.). The solubilization and denaturation of the harvested material were performed by resuspending the inclusion bodies in 100 mM Tris / HCl pH 8.0, 1 M EDTA, 100 mM DTT, 50 mM NaCl and 7 M urea and incubating at 37 ° C for 2 hours. After centrifugation to remove the insoluble material, the supernatant was extensively dialyzed in 50 mM MES pH 5.0, 0.1 mM EDTA, 10 mM DTT, and 7 M urea for cation exchange chromatography on the Resource S. column. The resulting combination of Fractions containing protease was then dialyzed in 25 mM Tris / HCl pH 7.8, 1 mM EDTA, and 1 M urea and diluted to approximately 100 μg / ml to which 0.2 mM oxidized glutathione and 2 mM reduced glutathione were added. Incubation was performed overnight at 4 ° C with gentle shaking and renaturation was then completed by intensive dialysis in 25 mM Tris / HCl pH 7.8, 1 mM EDTA, 1 mM DTT, and 100 'mM sodium chloride followed by dialysis in the storage buffer (20 mM acetate pH 5.0, 0.1 mM EDTA, 1 mM DTT, and 50 mM NaCl) and before the concentration of the sample to give the desired protease.
Example 2
ABz-Val-Val-Asn-Ala-Ser-Ser-Arg-Leu-Tyr (3-N02) -Arg-OH (1) [SEQ. ID. NO: 2]
Peptide 1 was assembled on the Wang resin of Na-Fmoc-NG-Pmc-L-Arginine (0.31 mmol / g, 0.2 mmol, 650 mg) using a Fmoc / DCC / HOBt solid phase procedure on a peptide synthesizer ACT396 MPS. The side chain protecting groups were P c for Arg, Trt for Asn, and O-tBu for Ser. The amino protecting groups, Fmoc, were removed in each step by washing the resin with NMP, stirring with a solution of 25 g. Piperidine% in NMP (3.5 ml for 5 minutes and then 3.5 ml for 20 minutes) and washing successively with NMP, methanol and NMP. The amino acids were coupled as their activated HOBt esters, using four equivalents of the reagents as follows: a 0.5 M solution of the amino acid protected with Fmoc and HOBt in NMP (1.6 ml, 0.8 mmol each) was automatically added to the unprotected resin , followed by the addition of a solution of 0.5 M DIC in NMP (1.6 ml, 0.8 mmol). The reaction vessel was stirred for 4 hours. The solution was then removed by filtration and the remaining resin was washed twice with 3.5 ml of NMP. Fresh portions of reagent solutions were added and the coupling repeated for 4 hours. At the end of each cycle, the resin was washed successively with NMP, methanol and NMP. The last coupling with N-Boc-2-aminobenzoic acid (Boc-ABz-OH) was carried out only with 1.1 equivalent of the reagents for 4 hours (simple coupling) in order to avoid the formation of the tyrosine esterification byproduct ( the hydroxyl of tyrosine is unprotected). The resulting resin was washed successively as described above, followed by further washing with methylene chloride, and then dried under reduced pressure. Complete deprotection and cleavage of the resin was achieved by shaking the resin (890 mg) with 10 ml of a mixture of TFA / anisole / thioanisole / ethanedithiol (90/2/3/5) for 2.5 hours at room temperature under nitrogen atmosphere. The mixture was filtered by suction in Et20 (200 ml) to give a yellow precipitate. This suspension was cooled in an ice bath and filtered through a 45 μm membrane to give a yellow solid (281 mg, 47% homogeneity by analytical HPLC). The crude product was purified in two batches (2x140 mg) by preparative HPLC (Whatman HPLC column, 22.0 mm x 500 mm, Partisil® 10 ODS-3 M / 20-50, particle size 10 μm, solvents: A = 0.06% TFA / H20, B = 75% CH3CN-25% H20 containing 0.06% TFA; gradient from 0 to 35% of B in 60 minutes). After lyophilization, compound 1 was recovered as an amorphous yellow solid (101.5 mg, 95% homogeneity by HPLC). This material was repurified on the same preparatory HPLC using a faster gradient of 0 to 40% B in 60 minutes. The product (62 mg, 96% homogeneity by HPLC) was purified a third time by preparative HPLC using an even faster solvent gradient (0 to 50% B in 60 minutes) to produce 31 mg (99% homogeneity per HPLC) of the desired peptide of formula 1. MS-FAB (thioglycerol): MH + = 1329.9 Da. Amino acid analysis: cale, (obs.) Asx 1 (0.99), Ser 2 (1.93),
Arg 2 (1.99), Wing 1 (1.01) ', Leu 1 (1.07), Val 2
(1.48, partial hydrolysis), Tyr (3-N02) 1 (present), 2-aminobenzoic acid (present), recovery of the peptide: 79% ± 6%.
Example 3
ABz-Tbg-Tbg-Asn (Me) 2-Ala-Ser-Ser-Arg-Leu-Tyr (3-N02; Arg-OH (2) [SEQ ID NO: 3]
This peptide was essentially synthesized as described above using a "Vega Biotechnologies Coupler® 250 C" agitator-like peptide synthesizer. A mixture of Fmoc-L-amino acid (1 mmol), HOBt (140 mg, 1 mmol) and 1 M solution of DCC in methylene chloride (1 mL, 1 mmol), NMP (2 mL) and methylene chloride ( 6 ml) was stirred at room temperature (20-22 ° C) for 30 minutes. The resulting suspension was filtered. The filtrate was diluted with NMP (10 ml) and added to the resin deprotected with Fmoc. The complete coupling was ensured by Kaiser tests performed on a resin sample. After the final coupling the peptide-resin was washed as described above and dried under reduced pressure. The residue was stirred with 10 ml of a mixture of TFA / anisole / thioanisole / ethanedithiol (90/2/3/5) for 4 hours under nitrogen atmosphere. The resulting mixture was poured into cold Et20 (200 ml) to give a yellow precipitate. After filtration, a yellow solid was recovered (450 mg, HPLC indicated the presence of two main components: 24%, 19.28 minutes and 34%, 21.88 minutes). The crude product (200 mg) was purified by preparative HPLC (Whatman HPLC column, 22.0 mm x 500 mm, Partisil® 10 0DS-3 M / 20-50, particle size 10 μm, solvents, A = 0.6% TFA / H20, B = 75% CH3CN-25% H20 containing 0.06% TFA, gradient: 10 to 40% B in 30 minutes, then 40% to 50% B in 30 minutes). After lyophilization a yellow compound of formula 2 was obtained as an amorphous solid (24 mg, homogeneity greater than 99% by HPLC). MS-FAB (thioglycerol): MH + = 1384.9 Da, amino acid analysis: cale. (obs) Asx 1 (1.03), Ser 2 (1.86), Arg 2 (2.02), Ala 1 (1.05), Leu 1 (1.05), Tbg 2 (1.12, partial hydrolysis), Tyr (3-N02) 1 (0.88), 2-aminobenzoic acid 1 (0.71), recovery of the peptide: 75.2% ± 0.6%.
Example 4
N-acetyl-Tbg-Tbg-Asn (Me) 2-Ala-AMC (3) [SEQ. ID. NO: 5]
This substrate was synthesized in solution using the following procedure: a) Boc-Al to -AMC (4). N, N-diisopropylethylamine (DIPEA, 3.6 ml, 20.5 mmol) was added to an ice-cooled solution of Boc-L-alanine (1.55 g, 8.20 mmol), 7-amino-4-methylcoumarin (MCA, 0.72 g, 4.1 mmol) and TBTU (2.63 g, 8.19 mmol) in DMF (10 ml). The mixture was stirred in an ice bath for 30 minutes under nitrogen atmosphere, then at room temperature in the dark for 15 hours. The reaction mixture was diluted with ethyl acetate (60 ml) and the solution was washed successively with 2N NaOH (3 x 30 ml), 1 N HCl (3 x 30 ml), water (3 x 20 ml) and saturated brine. (2 x 20 ml). The organic solution was then dried over sodium sulfate, filtered and concentrated under reduced pressure to give a white solid in residual ethyl acetate. This residue was triturated with Et20 (50 ml), filtered and rinsed successively with Et20 (3 x 20 ml) and hexane (3 x 20 ml) and dried under reduced pressure to give a white solid product (0.657 g, 46 g). % yield, 98% homogeneity by HPLC). MS-FAB: MH + = 347 Da; NMR * H (400 MHz, DMS0-d6) d 10.36 (s, HH), 7.76 (d, J = 1.6 Hz, HH), 7.72 (d, J = 8.6 Hz, HH), 7.50 (d, J = 8.6 Hz, ÍH), 7.16 (d, J = 6.7 Hz, ÍH), 6.25 (s, ÍH), 4.13 (m, J = 7.0 Hz, ÍH), 2.40 (s, 3H), s, 9H), 1.28 ( d, J = 6.7 Hz, 3H)
b) HCl • H-Al to -AMC (5). Boc-Ala-AMC (4) (0.557 g, 1.61 mmol) was dissolved in 4N HCl / dioxane solution
(30 ml). After 5 minutes of stirring, a white precipitate began to form. The mixture was stirred at room temperature for 45 minutes under a nitrogen atmosphere in the dark and then concentrated under reduced pressure to semi-dryness. The residual white paste was triturated with Et20 (50 ml), filtered and dried under reduced pressure to give compound 5 as a yellowish, off-white solid.
(0.495 g, quantitative yield, 96% homogeneity by HPLC). This product was not further characterized and was used directly in the next synthetic step.
c) Boc -Asn (Me)? -Al to -AMC (6). N, N-diisopropyl-ethylamine (DIPEA, 1.7 ml, 9.76 mmol) was added to a solution containing N ', N' -dimethyl-Boc-L-Asparagine (0.5 g, 1.92 mmol), crude compound 5 (presumably 1.61 mmol) and TBTU (0.617 g, 1.92 mmol) in dimethylformamide (5 ml). The mixture was stirred under nitrogen atmosphere at room temperature in the dark for 2.5 hours. The reaction mixture was then diluted with ethyl acetate (50 ml) and the solution was washed successively with 2N sodium hydroxide (3 x 20 ml), IN HCl (3 x 20 ml), water (2 x 20 ml) and saturated brine (2 x 20 ml). During the washes, the product was precipitated; it was redissolved by the addition of more EtOAc (30 ml). The organic solution was then dried over sodium sulfate, filtered and concentrated under reduced pressure to give a white semi-solid residue. This residue was triturated with Et20 (100 ml) and filtered through a 45 μm membrane. The resulting solid was rinsed successively with Et20 (3 x 20 ml) and hexane
(3 x 20 ml). After this, it was dried under reduced pressure to give a white solid product (0.470 g, 60% yield, 99% homogeneity by HPLC). MS-FAB: MH + = 489 Da, (M + Na +) = 511 Da, XH NMR (400 MHz, DMS0-d6) d 10.08 (s, HH), 8.27 (d, J = 7.0 Hz, HH), 7.82 ( d, J = 1.9 Hz, HH), 7.73 (d, J = 8.6 Hz, HH), 7.61 (dd, J = 8.6, 1.9 Hz, 1H), 6.96 (d, J = 7.6 Hz, HH), 6.26 ( d, J = 1.3 Hz, HH), 4.39 (m, HH), 4.34 (m, HH), 2.96 (s, 3H), 2.85 (s, 3H), 2.72 (d, J = 6.4 Hz, 2H), 2.40 (d, J = 0.9 Hz, 3H), 1.39 (s, 9H), 1.34 (d, J = 7 Hz, 3H).
d) HCl H-Asn (Me)? -Al to -AMC (7) Boc-Asn (Me2) -Ala-AMC (6) (0.370 g, 0.76 mmol) was deprotected in 2 hours using the standard procedure described for compound 5, resulting in recovery of the compound 7 (0.320 g, 100% yield, 99% homogeneity with HPLC). This product was not subsequently characterized but was used directly as such for the next step.
e) Boc-Tbg-Asn (Me) 2 -Al to -AMC (8). The N, N-diisopropylethylamine (DIPEA, 0.8 ml, 4.59 mmol) was added to a solution containing Boc-L-tert-but-il-glycine (Boc-Tbg-OH, 0.21 g, 0.9 mmol), the crude compound 7 (presumably 0.76 mmol) and TBTU (0.29 g, 0.9 mmol) in dimethylformamide (3.5 ml). Treatment of the mixture in the same manner as described for the corresponding mixture in the above preparation of compound 6 gave a white solid product (0.358 g, 79% yield, 99% homogeneity by HPLC). MS-FAB: MH + = 602 Da, XH NMR (400 MHz, DMSO-d6) d 9.96
(s, ÍH), 8.38 (d, J = 7.3 Hz, 1H), 8.15 (d, J = 7.3
Hz, ÍH), 7.86 (d, J = 1.9 Hz, 'ÍH), 7.74 (part of an AB system, d, J = 8.6 Hz, ÍH), 7.68 (part of an AB system, dd, J = 8.6, 1.9 Hz, ÍH), 6.53 (d, J = 8.9 Hz, ÍH), 6.27 (s, ÍH), 4.66 (m, ÍH), 4.38 (quintuplete, J = 7.3 Hz, ÍH), 3.87 (d, J = 9.2 Hz, HH), 2.98 (s, 3H), 2.86 (s, 3H), 2.85-2.70 (m, 2H), 2.40 (s, 3H), 1.39 (s, 9H), 1.33 (d, J = 7.3 Hz, 3H), 0.89 (s, 9H).
f) HCl • H- Tbg-Asn (Me) 2 -Al to -AMC (9). Boc-Tbg-Asn (Me) 2-Ala-AMC (8) was deprotected in 3 hours using the standard procedure described for compound 5. In this way, compound 9 was obtained (0.240 g, quantitative yield, 97% of homogeneity by HPLC). This product was not subsequently characterized but was used as such for the next step.
g) Boc - Tbg- Tbg-Asn (Me)? -Al to -AMC (10). N, N-diisopropylethylamine (DIPEA, 0.5 ml, 2.57 mmol) was added to a solution containing Boc-L-tert-butyl-glycine (Boc-Tbg-OH, 0.119 g, 0.515 mmol), the crude compound 9 (presumably 0.43 mmol) and TBTU (0.165 g, 0.515 mmol) in dimethylformamide (2 ml). The mixture was treated in the same manner as described for the corresponding mixture in the preparation of compound 6. In this way, a white solid was obtained (0.227 g, 74% yield, 96% homogeneity by HPLC). MS-FAB: MH + = 715 Da, NMR? H (400 MHz, DMSO-d6) d 9.95 (s, ÍH), 8.35 (d, J = 7.6 Hz, ÍH), 8.32 (d, J = 7.6 Hz, ÍH) ), 7.86 (d, J = 1.9 Hz, ÍH), 7.74 (part of an AB system, d, J = 8.6 Hz, ÍH), 7.68 (part of an AB system, dd, J = 8.6 Hz, 1.9 Hz, ÍH), 7.53 (d, J = 9.2 Hz, ÍH), 6.85 (d, J = 9.2 Hz, ÍH), 6.27 (d, J = 0.9 Hz, ÍH), 4.64 (m, ÍH), 4.39 (quintuplete, J = 7.3 Hz, 1H), 4.29 (d, J = 8.9 Hz, 1H), 3.93 (d, J = 9.5 Hz, ÍH), 2.97 (s, 3H), 2.86
(s, 3.5 H), 2.82 (part of an AB system, d, J = 8.9 Hz, 0.5H), 2.67 (part of an AB system, dd, J = 16.2, 5.1 Hz, ÍH), 2.40 (d, J = 0.9 Hz, 3H), 1.39 (s, 9H), 1.32 (d, J = 7.3 Hz, 3H), 0.911 (s, 9H), 0.907
(s, 9H).
h) HCl • H- Tbg-Tbg-Asn (Me) 2 -Al to -AMC (11). Boc-Tbg-Tbg-Asn (Me) 2-Ala-AMC (10) (0.127 g, 0.177 mmol) was deprotected in 2 hours using the standard procedure described for compound 5. Therefore, compound 11 was obtained ( 0.125 g, quantitative yield, 96% homogeneity by HPLC). This product was not further characterized but was used directly in the next step.
i) Ac - Tbg - Tbg - Asn (Me)? -Al to -AMC (3). Acetic anhydride (50 ml, 0.53 mmol) was added to a solution of crude compound 11 (presumably 0.177 mmol) in 1 ml of pyridine. The mixture was stirred at room temperature in the dark and under a nitrogen atmosphere for 1 hour. The reaction mixture was then diluted with ethyl acetate (30 ml). The solution was washed successively with IN HCl (3 x 20 ml), with water (3 x 20 ml), with saturated brine (2 x 20 ml), dried over sodium sulfate, filtered and concentrated under reduced pressure. The semi-solid residue was triturated with Et20 (100 ml) and filtered through a 45 μm membrane. The resulting solid was washed successively with Et20 (3 x 10 ml) and with hexane (3 x 10 ml) and dried under reduced pressure for 18 hours to give the substrate 3 as a white solid product (90 mg, 77% yield). , 98% homogeneity by HPLC). MS-FAB: MH + = 657 Da, NMR? E (400 MHz, DMS0-d6) d 9.95 (s, ÍH), 8.39 (d, J = 7.3 Hz, ÍH), 8.20 (d, J = 7.3 Hz, ), 7.86 (d, J = 1.9 Hz, ÍH), 7.82 (d, J = 9.2 Hz, ÍH), 7.73 (part of an AB system, d, J = 8.6 Hz, 1H), 7.67 (part of a system AB, dd, J = 8.6, 1.9 Hz, ÍH), 7.61 (d, J = 8.9 Hz, ÍH), 6.26 (d, J = 1.3 Hz, ÍH), 4.62 (m, ÍH), 4.37 (m, ÍH) ), 4.35 (d, J = 9.6 Hz, ÍH), 4.22 (d, J = 8.9 Hz, ÍH), 2.97 (s, 3H), 2.86 (s, 3H), 2.81 (part of an AB system, dd, J = 16.2, 8.6 Hz, ÍH), 2.70 (part of an AB system, d, J = 16.2, 4.1 Hz, ÍH), 2.40 (d, J = 1.3 Hz, 3H), 1.89 (s, 3H), 1.32 (d, J = 7.3 Hz, 3H), 0.907 (s, 9H), 0.904 (s, 9H). Amino acid analysis: cale. (obs.) Asx 1 (0.99), Ala 1 (1.01), Tbg 2 (1.15, partial hydrolysis), 7-amino-4-methylcoumarin 1 (1).
Example 5 d-Biotin-Arg-Gly-Val-Val-Asn-Ala-Ser-Ser-Arg-Leu-Ala-Tyr-NH2 (12) [SEQ. ID. NO 6]
This compound was synthesized by the use of an Applied Biosystem peptide synthesizer (Model 430 A) and following an Fmoc / DCC / HOBt protocol. The synthesis was performed on 0.25 mmol of Rink resin (Advanced Chemtech, Louisville KY, USA) with 1 mmol of protected amino acids and the coupling was allowed to proceed for 1 hour. The simple coupling was used for Tyr, Ala, Leu and Arg while the double coupling was used for Ser, Ser, Ala, Asn, Val, Gly and Arg. The biotin was coupled with TBTU in a 1: 3 mixture of DMSO / NMP for 24 hours. After the final coupling the peptide-resin was washed as described above and dried under reduced pressure. The cleavage and concomitant deprotection of the side chain was performed in 10 ml of a mixture of TFA / phenol / thionanisole / ethanedithiol / H20 (90 / 3.5 / 2.5 / 1.2 / 2.5) for 2.5 hours under a nitrogen atmosphere. The cleavage cocktail was concentrated under reduced pressure. The residue was diluted with 20 ml of Et20. the resulting precipitate was collected on a filter and purified by reverse phase semi-preparative HPLC (Whatman HPLC column, 22.0 mm x 500 mm, Partisil® 10 ODS-3 M / 20-50, particle size 10 μm, solvents : A = 0.06% of TFA / H20, B = 75% CH3CN-25% of H20 that contained 0.06% of TFA, gradient: 0 to 40% of B in 60 minutes). Fractions containing the desired product were combined, lyophilized and re-purified under the same conditions to provide, after lyophilization, the desired compound as a white amorphous solid (88 mg, 96% homogeneity by HPLC). MS-FAB (thioglycerol): MH + = 1517.6 Da, amino acid analysis: cale, (obs.) Asx 1 (0.97), Ser 2 (1.79), Gly 1 (1.03), Arg 2 (1.87), Ala 2 (2.06) ), Tyr 1 (0.98), Val 2 (1.23, partial hydrolysis), Leu 1 (1.09), recovery of the peptide: 68% ± 1%.
Example 6
d-biotin-Arg-Gly-Tbg-Tbg-Asn (Me) 2-Ala-Ser-Ser-Arg-Leu-Ala-Tyr-NH2 (13) [SEQ. ID. NO: 7]
This compound was prepared on an ACT396 MPS peptide synthesizer using four amino acid equivalents, HOBt hydrate and TBTU and eight equivalents of DIPEA. Each amino acid was double-coupled. The reaction time for each coupling was 1 hour. At the end of the synthesis, the biotin residue was incorporated using a protocol similar to that described above. The cleavage and concomitant deprotection of the side chain was performed in 10 ml of a mixture of TFA / anisole / thioanisole / ethanedithiol (90/3/5/2) for 2.5 hours under a nitrogen atmosphere. The cleavage cocktail was concentrated under reduced pressure. The residue was diluted with 20 ml of Et20. The resulting precipitate formed was filtered and purified by semi-preparative, reverse phase HPLC (Waters PrepPak 500 cartridge, 60 mm x 330 mm, C18, particle size 15 μm, solvents: A = 0.05 M NH OAc / H20, B = CH3CN; gradient: 0 to 35% B in 80 minutes; flow rate: 50 ml / minute). The fractions containing the product were combined, lyophilized and re-purified (Water PrepPak 500 cartridge, 60 mm x 330 mm, C18, particle size 15 μm, solvents: A = 0.06% TFA / H20, B = 75% CH3CN-25% H20 containing 0.06% TFA, gradient: 0 to 40% B in 90 minutes, flow rate: 50 ml / minute). After lyophilization, the desired compound was isolated as a white amorphous solid (60 mg, 95% homogeneity by HPLC). MS-FAB
(thioglycerol): MH + = 1573.8 Da, amino acid analysis: cale, (obs.) Asx 1 (1.02), Ser 2 (1.84), Arg 2 (2.01), Ala 2 (2.05), Tyr 1 (1.00), Leu 1
(1.05), Tbg 2 (0.25, partial hydrolysis), recovery of the peptide: 66% ± 2%.
Dialing with 125t (13a)
Marking with 125 e biotin-Arg-Gly-Tbg-Tbg-Asn (NMe) 2-Ala ^ Ser-Ser-Arg-Leu-Ala-Tyr-NH2 (13).
Substrate 13 was labeled with Na125I using the insoluble iodination reagent 1, 3, 4,6-tetrachloro-3a, 6a-diphenylglyceryl (IODO-GEN® from Pierce) as instructed by the manufacturer. In brief, 250 μl of a 100 μg / ml solution of IODO-GEN® in chloroform was slowly evaporated with a stream of nitrogen in a borosilicate tube of 16 × 100 mm. To the tube were added 187 μl of 0.5 M potassium phosphate buffer cooled with ice, pH 7, and 13 μl of 1 mM 13 substrate in DMSO. Iodization was initiated by the addition of 10 μl of Na125I 1 mCi. The reaction was allowed to proceed for 45 seconds at 0 ° C, after which time 300 μl of 20% aqueous acetonitrile + 0.1% trifluoroacetic acid were added. The reaction mixture was immediately injected onto a 25 cm Vydac C18 reversed phase column, equilibrated with 20% aqueous acetonitrile + 0.1% trifluoroacetic acid. The column was washed under the same conditions for 10 minutes, followed by a linear elution gradient (20-80% aqueous acetonitrile + 0.1% trifluoroacetic acid) in 30 minutes. After the appearance of the peak corresponding to the unreacted substrate 13, the 250 μl fractions were immediately collected and counted. The first peak of radioactivity after substrate 13 corresponded to monabeled substrate 13a (biotin-Arg-Gly-Tbg-Tbg-Asn (NMe) 2-Ala ^ Ser-Ser-Arg-Leu-Ala-Tyr (125I) -NH2 ). These fractions were combined and the excess acetonitrile was evaporated. Casein, cold substrate 13 and dithiothreitol were added to the combined, at a final concentration of 0.1% w / v, 20 μM and 5 mv to prevent adhesion and oxidation problems. The 13a substrate solution was stored in aliquots at 4 ° C and was stable for 3 to 4 weeks.
Example 7
Ac-Tbg-Tbg-Asn (Me) 2-Ala-pNP [SEQ. ID. NO: 8]
Pure P0C13 (1.0 mL, 11 mmol) was added at -15 ° C in 2 minutes to a solution of Boc-Ala-OH (1.89 g, 10 mmol) and p-nitroaniline (1.38 g, 10 mL) in pyridine (30 mL). ml). The mixture was stirred for 30 minutes and then emptied into 100 ml of ice water and the resulting mixture was extracted with ethyl acetate (3 x 50 ml). The organic layers were combined and washed successively with 1M HCl (3 x 50 ml), with saturated aqueous sodium bicarbonate (3 x 50 ml) and saturated brine (3 x 50 ml). The organic layer was dried over sodium sulfate and the solvent was evaporated under reduced pressure. The residual solid was triturated with ether and filtered to give Boc-Ala-pNA as a yellow solid (1.72 g, 56% yield). NMR - "" H (DMSO-d6)? 10.53 (s, ÍH), 8.22 (d, J = 10.5 Hz, 2H), 7.85 (d, J = 9.2 Hz, 2H), 7.18 (broad d, J = 6.3 Hz, ÍH), 4.14 (t, J = 7.0 Hz, ÍH), 1.38 (s, 9H), 1.25 (d, J = 7.0 Hz, 3H). Beginning from Boc-Ala-pNA and using a sequential deprotection and coupling protocol as described for Example 4, the title compound was prepared as a white amorphous solid (after purification by semi-preparative HPLC). Electrorrocio-MS: MH + = 620.5 Da, Analysis of amino acids: cale, (obs.) Asx 1 (0.96), Ala 1 (1.04), Tbg 2 (present, partial hydrolysis), recovery of the peptide: 102 ± 1%.
Example
Ac-Tbg-Tbg-Asn (Me) 2-Ala-pNA [SEQ. ID. NO: 9]
The Ac-Tbg-Tbg-Asn (Me) 2-Ala-OBn precursor was synthesized in solution by an analogous process as that described in Example 4. The benzyl ester was hydrogenolyzed using a similar procedure as in Example 1, Step B Starting from Ac-Tbg-Tbg-Asn (Me) 2-Ala-OH, the title compound was prepared according to the following protocol: The above tetrapeptide (200 mg, 0.4 mmol) and p-nitrophenol (60 mg , 0.43 mmol) were dissolved in 2 ml of dimethylformamide and 5 ml of ethyl acetate. The mixture was cooled to 0 ° C and a solution of DCC (80 mg, 0.388 mmol) in 0.5 mL of ethyl acetate was added. The mixture was stirred at 0 ° C for 40 minutes, and then at room temperature overnight. The mixture was filtered, the filtrate was diluted with ethyl acetate and washed with saturated aqueous brine (2 x 10 ml), water (3 x 10 ml) and dried over sodium sulfate to give, after evaporation of the solvent , 214 mg of a crude material which showed two peaks of almost equal intensity by reverse phase HPLC. An attempt to purify the compound by chromatography on silica gel was performed but separation did not take place. All fractions were combined and purified by semi-preparative HPLC (same chromatographic column and same eluents as for Example 3). Gradient: 0 to 20% eluent B / A in 10 minutes, then 20 to 60% eluent B / A in 80 minutes. Of the two compounds that were isolated, the one that moved slowest (A), was the desired compound (white amorphous solid, 40 mg), while the compound (B) that moved faster was its corresponding diastereoisomer D-Ala (as determined on the digested peptide using a chiral GC column). Compound A: 100% homogeneity by HPLC, electro-dew-MS: MH + = 621.4 Da, amino acid analysis: cale. (obs.) Asx 1 (0.98), Wing 1 (1.02). Tbg 2 (1.29, partial hydrolysis), recovery of the peptide: 90 ± 8%.
Example 9
Ketoamide inhibitor based on peptide DA-Tbg-Tbg-Asn (Me2) -Ala-CONHCH2Ph [SEQ. ID. NO: 4]
To a solution of nitroethane (4.0 g, 53 mmol) in 15 ml of ethanol was added aqueous sodium hydroxide (68 ml of 2N solution, 136 mmol). To this rapidly stirred solution was added glyoxylic acid (5.9 g, 64 mmol). The solution was stirred 15 hours and then acidified with 10% aqueous hydrochloric acid (pH 2) and the aqueous phase was saturated with sodium chloride before extraction with ethyl acetate (3 x 150 ml). The organic phase was dried over magnesium sulfate, filtered and concentrated to give 8.1 g of a viscous yellow oil. This crude material was dissolved in 50 ml of ethanol containing Et3N (18 ml, 119 mmol) and treated with di-tert-butyl dicarbonate (12.2 g, 56 mmol) and Raney nickel (3 g), which had Was previously washed with water before use. Hydrogenation at 3.16 kg / cm2 (45 psi) for 20 hours provided, after filtration, through diatomaceous earth and concentration, the crude acid (11.1 g). A portion of the crude acid (3.07 g, 14 mmol) was dissolved in 30 ml of dimethylformamide and treated with anhydrous potassium carbonate (4.3 g, 30.8 mmol) and benzyl bromide (2.5 ml, 21 mmol). After stirring for 3 hours at room temperature, the DMF was removed under reduced pressure and the residue was dissolved in 150 ml of ethyl acetate and washed with 100 ml of water and 80 ml of brine. The organic phase was dried over magnesium sulfate, filtered and concentrated. The crude yellow oil (4.3 g) was purified by flash chromatography on silica gel (230-400 mesh) eluting with 33% ethyl acetate in hexane to give the pure benzyl ester BocNHCH (Me) CHOHC (O) 0-CH2Ph (1.8 g, 42% from nitroethane). HPLC (system C) 99%,
(system D) 97%; IR (KBr)? 3422, 3361, 1740, 1684 cm "1; XH NMR (400 MHz, CDC13) d 7.36 (s, 5H), 5.27 (d, J = 12.1 Hz, ÍH), 5.19 (d, J = 12.1 Hz, ÍH) , 4.82 (m, 1H), 4.36 and 4.35 (2 xd, J = 5.7 and 5.4 Hz, 1H), 4.11
(m, ÍH), 3.10 (m, ÍH), 1.43 (s, 9H), 0.97 (d, J = 7.0
Hz, 3H); FAB MS m / z: 310 (MH +), 210 (M-100); HRMS calculated for Ci6H24N05 (MH +) 310.1654, found: 310.1644. The tert-butyloxycarbonyl (Boc) group from the preceding benzyl ester (4.0 g, 12.9 mmol) was removed using 4 N HCl / dioxane (30 ml) for 45 minutes at 0 ° C. The hydrochloride salt was obtained by concentration and coevaporation with 15 ml of toluene. The hydrochloride salt (12.9 mmol) was combined with l-ethyl-3- (3-dimethylaminopropyl) carbodiimide (2.6 g, 13.6 mmol, 1.1 equivalent), HOBT (1.8 g, 13.6 mmol, 1.1 equivalent) and Boc-Asn ( NMe2) -OH (3.4 g, 12.9 mmol, 1.1 equivalent) in 50 ml of dimethylformamide under a nitrogen atmosphere. The solution was cooled to 0 ° C (ice bath) before iPr2NEt (7.9 ml, 45.3 mmol, 3.5 equivalents) was added. The solution was then stirred at room temperature for 16 hours. The reaction mixture was partitioned between 250 ml of ethyl acetate and saturated aqueous sodium bicarbonate (150 ml). The organic phase was washed with 150 ml of 5% aqueous HCl and finally with 150 ml of brine. It was dried over magnesium sulfate, followed by filtration and concentration to give 6.0 g of the crude material. In most cases the raw material was suitable for subsequent couplings without purification. After the final coupling, the peptide of the -hydroxybenzyl ester was purified by flash chromatography. The corresponding α-hydroxy acid was then obtained from the benzyl ester (1.10 g, 2.0 mmol) by hydrogenation over 10% Pd / C (55 mg) in 30 ml of ethanol at atmospheric pressure over the course of a few hours, for provide after filtration through a pad of diatomaceous earth, a white solid (0.95 g, 100% yield). HPLC (system A) 100%, (system C) 100%; IR (KBr)? 3316, 1727, 1642 cm "1; XH NMR (400 MHz, CDCl3), mixture of 4 diastereoisomers, d 8.06 and 8.01 (2 xd, J = 7.3 and 8.6 Hz, ÍH), 7.87, 7.79, 7.70 and 7.54 (4 xd, J = 8.6, 8.6, 8.9 and 8.6 Hz, ÍH), 7.09 and 7.03 (2 xd, J = 7.9 and 8.6 Hz, 0.5H), 6.72 (m, 0.5H), 6.52 (m, 0.25), 6.34 and 6.29 (2 xd, J = 7.6 and 7.3 Hz, 0.75H), 6.10-5.4 (s broad, ÍH), 4.99-4.88 (m, 0.5H), 4.87-4.78 (m, 0.5H), 4.66-4.37 (m, 2H), 4.33-4.09 (m, ÍH), 3.30-3.15 (m, 0.3H), 3.05-2.85 (, 6.7H), 2.75-2.65 and 2.60-2.50 (m, ÍH), 2.25-2.10 (m, 2H), 1.28-1.19 (m, 3H), 1.10-0.97 (m, 18H); 13C NMR (100.6 MHz, CDC13) d 174.9, 173.5, 173.1, 173.0, 171.5, 171.0, 170.9, 170.8, 170.7 , 170.5, 170.2, 73.03, 72.74, 60.9, 60.67, 50.3, 50.1, 49.5, 49.4, 48.1, 47.9, 47.7, 37.56, 35.9, 35.8, 35.7, 34.7, 34.4, 34.3, 33.8, 31.1, 29.9, 29.8, 26.9 , 26.8, 26.7, 17.4, 17.2; FAB MS m / z: 473 (MH +), 495 (M + 23) The coupling of the Pi residue 'was achieved using the general coupling protocol described above with benzylamine (1.2 equivalents). The final oxidation step was performed by treatment of the pre-requisite a-hydroxyamide (62 mg, 0.11 mmol) with 2 equivalents of 1,1-triacetoxy-1, 1-dihydro-1,2-benzyodoxol-3 (1H). -one (94 mg, 0.22 mmol) in 1 ml of dimethylformamide for 4 hours. Addition of 5 ml of 10% sodium thiosulfate and 5 ml of saturated sodium bicarbonate, with stirring for 15 minutes, was followed by extraction with ethyl acetate (3 x 10 ml) to give the desired a-ketoamide. The final purification was performed using preparative HPLC to provide, after lyophilization, DATbg-Tbg-Asn (Me2) -Ala-C (0) NHCH2Ph, (51 mg, 82% yield) as a white solid. HPLC (system C) 100%,
(system D) 96.1%; IR (KBr)? 3316, 1641, 1529 cm "1; 1 H NMR (400 MHz, DMSO-d6), mixture of diastereomers 1: 1 to Pi, d 9.21-9.15 (m, HH), 8.14 and 8.09 (2 xd, 7.3 and 7.6 Hz , ÍH), 8.03 and 7.97 (2 xd, J = 6.4 and 5.7 Hz, ÍH), 7.60 (d, J = 8.3, HH), 7.35-7.17 (m, 5H), 5.02-4.88 (m, 1H), 4.64-4.49 (m, ÍH), 4.39-4.23 (m, 2H), 4.13 and 4.12 (2 xd, J = 8.6 and 8.6 Hz, ÍH), 2.92 and 2.91 (2 xs, 3H), 2.79 and 2.78 (2 xs, 3H), 2.74-2.54 (, 2H), 2.19 (broad d, J = 12.4 Hz, ÍH), 2.03 and 2.02 (2 xd, J = 12.4 and 12.7 Hz, ÍH), 1.25 and 1.23 (2 xd, J = 7.3 and 7.0 Hz, 3H), 0.94 and 0.91 (2 xs, 18H); FAB MS m / z: 560 (MH +), 582 (M + 23).
Example 10
HCMV protease assay
The stock solutions (10 mM) in DMSO of the compounds prepared in Examples 2, 3, 4, 5 and 6 were stored at 4 ° C. The compounds in these solutions were stable for several weeks under these conditions. Samples were taken as aliquots of the 50 μM HCMV protease (1.4 mg / ml) and kept at -80 ° C in storage buffer without detectable loss of activity over a prolonged period of more than one year. The calculated molecular mass of the wild-type HCMV protease was 28042 Da. The concentration of enzyme on which the calculations of the number of turns, kcat, and the specificity constant, kcat / Km, were derived was expressed in terms of the total concentration of monomer. HPLC analysis. The specificity constant, kcat / Km, and the individual kcat "and Km parameters for the peptide substrate of Example 2, were determined by HPLC by periodically checking the initial rate of occurrence of the N-terminal cleavage product at various substrate concentrations. Incubation buffer contained 50 mM Tris / HCl pH 8, 0.5 M sodium sulfate, 50 mM sodium chloride, 0.1 M EDTA, 1 mM TCEP, 3% DMSO v / v, and 0.05% w / v casein to which 20-500 μM substrate was added.After equilibration at 30 ° C, the reactions were initiated by the addition of 50 nM HCMV protease.
(total monomer concentration) from a 2.5 μM stock solution in assay buffer, and quenched at regular intervals with 1% TFA to ca. 25% total hydrolysis. The aliquots were injected into a C8 reversed phase column using a linear gradient of acetonitrile in 3 mM aqueous SDS containing 0.05% H3P04 (22 to 28% acetonitrile at 2 minutes, up to 30% in 4 minutes, up to 50% in 6 minutes at a constant flow rate of 4 ml / minute). A calibration curve of the N-terminal product was used to estimate the degree of conversion (retention time = 2.7 minutes). The data were adjusted to the non-linear regression analysis of the Michaelis-Menten equation using the computer software (software) kinetics GRAFIT (Grafit, Erithacus Software Ltd, 1989-1992, Version 3.0, Leatherbarrow, RJ, London, United Kingdom) . A similar protocol was adopted for the peptide substrates of Examples 5 and 6. In this case, the enzyme concentration used was respectively 500 nM and 200 nM. The HPLC gradient was from 25 to 38% acetonitrile at 11 minutes, up to 100% in 13 minutes at a constant flow rate of 4 ml / minute. Fl uoromé tri co test. Specificity constant, kcat / Km, and individual kcat and Km parameters for the peptide substrates of Examples 3 and 4, were determined by the initial rate kinetics as measured from the increase in fluorescence intensity after excision of the Amide bond Ala-Ser or Ala-AMC in the presence of 50 nM HCMV protease (for anthranilamide? ex = 312 nm? em = 415 n, for 7-amino-4-methylcoumarin? ex = 360 nm and? em = 440 nm The slot width sizes varied from 2.5 nm to 5 nm). The substrate concentrations above and below Km were used to accurately determine the individual kinetic parameters. The reaction conditions and the adjustment methods were as described above. Radiometric test: The utility of this optimized sequence Tbg-Tbg-Asn (Me) 2 was not limited to fluorogenic substrates and could be successfully applied to other methodologies. Of course, the introduction of this sequence into the radiometric substrate having the structure of formula 12a.
d-biotin-Arg-Gly-Val-Val-Asn-Ala-Ser-Ser-Arg-Leu-Ala-Tyr (125 I) -NH2 (12a) [SEQ. ID. NO 6]
led to the substrate having the structure of formula 13a:
d-biotin-Arg-Gly-Tbg-Tbg-Asn (Me) 2-Ala-Ser-Ser-Arg-Leu-Ala-Tyr (1l 25oI) -NH2 (13a) I SEQ. ID. NO: 7] whose kcat and KM values of 0.045 s "1 and 19 μM respectively, led to a specificity constant, kcat / KM, of 2370 M ^ s-1 for example 12 times higher compared to the substrate of the formula 12a (as measured by HPLC) These kinetic improvements allowed the conversion of a standard radiometric assay to a Scintillation Proximity Assay (Bosworth, N., Towers, P. (1989) Na ture 341, 167-8; and Brown, A.M., George, S.M., Blu e, A.J., Dushin, R.G., Jacobsen, J.S., Sonneberg-Reines, J. (1994) Anal. Bi ochem. 217, 139-147) with the considerable advantages of reducing enzyme concentration, incubation time and manipulations. This assay, based on 125 I, compares favorably to the already published HCMV SPA protease, which requires a longer substrate and the use of protein kinase A for phosphorylation with 33P (Baum, EZ, Johnston, SH, Bebernitz, GA, Gluzman, Y. (1996), Anal. Bi ochem. 231, 129-134). Cro-genetic assay: The kinetic parameters (kcat, Km and kcat / Km) for the colorimetric peptide substrates of Examples 7 and 8 were determined by the initial velocity kinetics as measured from the increase in absorbance a? = 405 nm after cleavage of the p-nitrophenol or p-nitroaniline portion in the presence of the HCMV protease. The substrate concentrations above and below Km can be used to accurately determine the individual kinetic parameters. The reaction conditions and the adjent data are as described above. Hence, the synthesis and characterization of the improved substrates for the HCMV protease have been described. The improvements were based on the replacement of the P-P2-Val-Val-Asn sequence of the previous substrates by the modified amino acids Tbg-Tbg-Asn (Me) identified during the QSAR studies of the inhibitors. These modifications shown to significantly increase the potency of the peptide-based inhibitors, were successfully transposed to the substrates and translated into lower Km and faster, second order cleavage rates. These improved substrates (the substrate of formula 2 and the substrate of formula 3) led to the development of low nanomolar enzymatic assays for the unambiguous characterization of potent HCMV protease inhibitors and the expansion of kinetic fluorescence applications, not possible with the fluorogenic substrates currently available. The kinetic characterization of HCMV protease mutants that show lower activity than the wild type enzyme is expected to be also greatly increased. Although the absolute kinetic parameters may vary depending on the enzyme preparation and the experimental conditions, the relative amounts described in this work are likely to be conserved. This strategy can also be successfully applied to other detection methods. An improved HPLC, the radiometric and chromogenic HCMV protease substrate (the substrate of formula 13a) has been prepared which allows rapid and convenient assays, including the scintillation proximity method. (Bosworth, N. et al., See below, Brown, A.M. and collaborators, see below, and Baum, E.Z. and collaborators, see below).
LIST OF SEQUENCES
1. GENERAL INFORMATION
APPLICANT: (A) NAME: BOEHRINGER INGELHEIM (CA ADÁ)
LTD, (B) STREET: 2100 CUNARD STREET (C) CITY: LAVAL (D) STATE: QUEBEC (E) COUNTRY: CANADA (F) POSTAL CODE. H7S 2G5 (G) TELEPHONE: (514) 682-4640 (H) TELEFAX: (514) 682-8434
ii) TITLE OF THE INVENTION: IMPROVED SUBSTRATES FOR THE PROTEASE OF HUMAN CITOMEGALOVIRUS
iii) SEQUENCE NUMBER: 9
iv) COMPUTER LEGIBLE FORM: A) TYPE OF MEDIUM: Flexible disk B) COMPUTER: PC compatible with IBM C) OPERATING SYSTEM: PC-DOS / MS-DOS D) SOFTWARE: Patentln Relay # 1.0, Version # 1.30 (EPO)
v) DATA OF THE CURRENT APPLICATION NUMBER OF THE APPLICATION: vi) DATA OF THE PREVIOUS APPLICATION: A) NUMBER OF THE APPLICATION: US 60 / 033,854 B) DATE OF SUBMISSION: 27-DEC-1996
INFORMATION FOR THE SEQUENCE: SEQ ID No
i) CHARACTERISTICS OF THE SEQUENCE: A) LENGTH: 4 amino acids B) TYPE: amino acid C) TYPE OF HEBRA: simple D) TOPOLOGY: linear
ii) TYPE OF MOLECULE: peptide
ix) CHARACTERISTICS: A) NAME / KEY: modified site B) POSITION: 1 D) OTHER INFORMATION: / marker = X / note = "X is a detectable marker, affinity marker or N-terminal capping group; is a sequence of amino acids, sufficient for the recognition of the substrate bound to it "
ix) CHARACTERISTICS: A) NAME / KEY: modified site B) POSITION: 1 D) OTHER INFORMATION: / note = "Xaa in position 1 is Tbg"
ix) CHARACTERISTICS: A) NAME / KEY: modified site B) POSITION: 2 D) OTHER INFORMATION: / note- "Xaa in position 2 is Tbg"
ix) CHARACTERISTICS: A) NAME / KEY: modified site B) POSITION: 3 D) OTHER INFORMATION: / note = "Xaa in position 3 is a divalent radical NHCH {CH2C (O) N (Ri) (R2) .}. -C (O) where Ri and R2 are hydrogen, alkyl, cycloalkyl ... or Ri and R2 form pyrrolidino, piperidino or morpholino "ix) CHARACTERISTICS: A) NAME / KEY: modified site B) POSITION: 4 D) OTHER INFORMATION: / Z-marker / note = "Z is an amino acid sequence or derivative thereof sufficient for recognition of the substrate and to which an affinity tag or a detectable tag is coupled, or Z is amino aromatic. .. "
xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 1: Xaa Xaa Xaa Ala 1
2) INFORMATION FOR THE SEQUENCE: SEQ ID No. 2:
i) CHARACTERISTICS OF THE SEQUENCE: A) LENGTH: 11 amino acids B) TYPE: amino acid C) TYPE OF HEBRA: simple D) TOPOLOGY: linear
ii) TYPE OF MOLECULE: peptide
ix) CHARACTERISTICS A) NAME / KEY: modified site B) POSITION: 1 D) OTHER INFORMATION: / product- "OTHER" / note- "Xaa in position 1 is aminobenzoic acid"
ix) CHARACTERISTICS: A) NAME / KEY: modified site B) POSITION: 9 D) OTHER INFORMATION: / product- "OTHER" / note- "Xaa in position 9 is 3-nitrotyrosine"
xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 2: Xaa Val Val Asn Ala Ser Ser Arg Leu Xaa Arg 1 5 10
2) INFORMATION FOR THE SEQUENCE: SEQ ID No
i) CHARACTERISTICS OF THE SEQUENCE: A) LENGTH: 11 amino acids B) TYPE: amino acid C) TYPE OF HEBRA: simple D) TOPOLOGY: linear ii) TYPE OF MOLECULE: peptide
ix) CHARACTERISTICS: A) NAME / KEY: modified site B) POSITION: 1 D) OTHER INFORMATION: / product- "OTHER" / note- "Xaa in position 1 is aminobenzoic acid"
ix) CHARACTERISTICS: A) NAME / KEY: modified site B) POSITION: 2 D) OTHER INFORMATION: / product- "OTHER" / note- "Xaa in position 2 is Tbg"
ix) CHARACTERISTICS: A) NAME / KEY: modified site B) POSITION: 3 D) OTHER INFORMATION: / product- "OTHER" / note- "Xaa in position 3 is Tbg"
ix) CHARACTERISTICS: A) NAME / KEY: modified site B) POSITION: 4 D) OTHER INFORMATION: / product- "OTHER" / note- "Xaa in position 4 is dimetil-asparagma"
ix) CHARACTERISTICS: A) NAME / KEY: modified site B) POSITION: 10 D) OTHER INFORMATION: / product- "OTHER" / note- "Xaa in position 10 is 3-nitrotyrosine"
Xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 3: Xaa Xaa Xaa Xaa Ala Ser Ser Arg Leu Xaa Arg 1 5 10
2) INFORMATION FOR THE SEQUENCE: SEQ ID No. 4:
CHARACTERISTICS OF THE SEQUENCE: A) LENGTH: 4 amino acids B) TYPE: amino acid C) TYPE OF HEBRA: simple D) TOPOLOGY: linear
ii) TYPE OF MOLECULE: peptide ix) CHARACTERISTICS: A) NAME / KEY: modified site B) POSITION: 1 D) OTHER INFORMATION: / note- "Xaa in position 1 is des-amino Tbg"
ix) CHARACTERISTICS: A) NAME / KEY: modified site B) POSITION: 2 D) OTHER INFORMATION: / note- "Xaa in position 2 is Tbg"
ix) CHARACTERISTICS: A) NAME / KEY: modified site B) POSITION: 3 D) OTHER INFORMATION: / note- "Xaa in position 3 is dimetilasparagine"
- ix) CHARACTERISTICS: A) NAME / KEY: modified site B) POSITION: 4 D) OTHER INFORMATION: / note- "COOH at the C-terminus is replaced by CONHCH2phenyl" xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO Xaa Xaa Xaa At 1
2) INFORMATION FOR THE SEQUENCE: SEQ ID No
CHARACTERISTICS OF THE SEQUENCE: A) LENGTH: 4 amino acids B) TYPE: amino acid C) TYPE OF HEBRA: simple D) TOPOLOGY: linear
ii) TYPE OF MOLECULE: peptide
ix) CHARACTERISTICS: A) NAME / KEY: modified site B) POSITION: 1 C) OTHER INFORMATION: '/ product- "N-terminal is encasquetado" / note- "the N-terminal amino group is encapsulated with acetyl"
ix) CHARACTERISTICS: A) NAME / KEY: modified site B) POSITION: 1 D) OTHER INFORMATION: / product- "OTHER" / note- "Xaa in position 1 is Tbg '
ix) CHARACTERISTICS: A) NAME / KEY: modified site B) POSITION: 2 D) OTHER INFORMATION: / product- "OTHER" / note- "Xaa in position 2 is Tbg '
ix) CHARACTERISTICS: A) NAME / KEY: modified site B) POSITION: 3 D) OTHER INFORMATION: / product- "OTHER" / note- "Xaa in position 3 is dimethyl-asparagma"
ix) CHARACTERISTICS: A) NAME / KEY: modified site B) POSITION: 4 D) OTHER INFORMATION: / marker- Z / note- "Z marker = aminomethylcoumarin (AMC)
xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO Xaa Xaa Xaa Ala 1 2) INFORMATION FOR THE SEQUENCE: SEQ ID No. 6
i) CHARACTERISTICS OF THE SEQUENCE: A) LENGTH: 12 amino acids B) TYPE: amino acid C) TYPE OF HEBRA: simple D) TOPOLOGY: linear
ii) TYPE OF MOLECULE: peptide
ix) CHARACTERISTICS: A) NAME / KEY: modified site B) POSITION: 1 D) OTHER INFORMATION: / marker- X / note- "X-marker is biotin"
ix) CHARACTERISTICS: A) NAME / KEY: modified site B) POSITION: 12 D) OTHER INFORMATION: / product- "C-terminal is replaced" / note- "the carboxyl group at the C-terminus is replaced by NH2"
xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO Arg Gly Val Val Asn Ala Ser Ser Arg Leu Ala Tyr 1 5 10
2) INFORMATION FOR THE SEQUENCE: SEQ ID No. 7
i) CHARACTERISTICS OF THE SEQUENCE: A) LENGTH: 12 amino acids B) TYPE: amino acid C) TYPE OF HEBRA: simple D) TOPOLOGY: linear
ii) TYPE OF MOLECULE: peptide
ix) CHARACTERISTICS: A) NAME / KEY: modified site B) POSITION: 1 D) OTHER INFORMATION: / marker- X / note- "X = d-biotin"
ix) CHARACTERISTICS: A) NAME / KEY: modified site B) POSITION: 3 D) OTHER INFORMATION: / note- "Xaa in position 3 is Tbg" ix) FEATURES: A) NAME / KEY: modified site B) POSITION: 4 D) OTHER INFORMATION: / note- "Xaa in position 4 is Tbg"
ix) CHARACTERISTICS: A) NAME / KEY: modified site B) POSITION: 5 D) OTHER INFORMATION: / note- "Xaa in position 5 is dimethyl-asparagine"
ix) CHARACTERISTICS: A) NAME / KEY: modified site B) POSITION: 12 D) OTHER INFORMATION: / note- "COOH at the C-end is replaced by NH2"
xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 7: Arg Gly Xaa Xaa Xaa Ala Ser Ser Arg Leu Ala Tyr 1 5 10
2) INFORMATION FOR THE SEQUENCE: SEQ ID No. 8
i) CHARACTERISTICS OF THE SEQUENCE:
A) LENGTH: 4 amino acids B) TYPE: amino acid C) TYPE OF HEBRA: simple D) TOPOLOGY: linear
ii) TYPE OF MOLECULE: peptide
ix) CHARACTERISTICS: A) NAME / KEY: modified site B) POSITION: 1 D) OTHER INFORMATION: / product- "N end is encasquetado / note-" the amino group at the N-end is encased with acetyl "
ix) CHARACTERISTICS: A) NAME / KEY: modified site B) POSITION: 1 D) OTHER INFORMATION: / product- "OTHER" / note- "Xaa in position 1 is Tbg"
ix) CHARACTERISTICS: A) NAME / KEY: modified site B) POSITION: 2 D) OTHER INFORMATION: / product- "OTHER" / note- "Xaa in position 2 is Tbg"
ix) CHARACTERISTICS: A) NAME / KEY: modified site B) POSITION: 3 D) OTHER INFORMATION: / product- "OTHER" / note- "Xaa in position 3 is dimetil-asparagma"
ix) CHARACTERISTICS: A) NAME / KEY: modified site B) POSITION: 4 D) OTHER INFORMATION: / marker-Z / note- "Z-marker = para-nitrophenol (pNP)"
xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 8 Xaa Xaa Xaa Ala 1
2) INFORMATION FOR THE SEQUENCE: SEQ ID No
CHARACTERISTICS OF THE SEQUENCE: A) LENGTH: 4 amino acids B) TYPE: amino acid C) TYPE OF HEBRA: simple D) TOPOLOGY: linear
ii) TYPE OF MOLECULE: peptide
ix) CHARACTERISTICS: A) NAME / KEY: modified site B) POSITION: 1 D) OTHER INFORMATION: / product- "N end is encasquetado" / note- "the N-terminal amino group is encapsulated with acetyl"
ix) CHARACTERISTICS: A) NAME / KEY: modified site B) POSITION: 1 D) OTHER INFORMATION: / product- "OTHER" / note- "Xaa in position 1 is Tbg '
ix) CHARACTERISTICS: A) NAME / KEY: modified site B) POSITION: 2 D) OTHER INFORMATION: / product- "OTHER" / note- "Xaa in position 2 is Tbg 'ix) FEATURES: A) NAME / KEY: modified site B) POSITION: 3 D) OTHER INFORMATION: / product- "OTHER" / note- "Xaa in position 3 is dimethyl-asparagma"
ix) CHARACTERISTICS: A) NAME / KEY: modified site B) POSITION: 4 D) OTHER INFORMATION: / marker-Z / note- "Z-marker = para-nitroaniline
(pNA)
xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 9: Xaa Xaa Xaa Ala 1
It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.
Claims (27)
1. A substrate, suitable for cleavage by a herpesvirus protease, represented by formula I X-Tbg-Tbg-Y-Ala-Z (I) [SEQ. ID. NO: 1] wherein X is a fluorescent donor moiety, a fluorescent acceptor moiety, an affinity tag or label, a detectable marker or an N-terminal capping group, or X is an amino acid sequence or derivative thereof, sufficient for recognition of the substrate to which a fluorescent donor moiety, a fluorescent acceptor moiety, an affinity tag, a detectable tag, or an N-terminal casing group is optionally coupled; And it's NHCH. { CH2C (O) N (R1) (R2)} C (0) wherein R1 and R2 are independently selected from hydrogen, alkyl of 1 to 6 carbon atoms, lower cycloalkyl, or (lower cycloalkyl) - (alkyl of 1 to 6 carbon atoms) or R1 and R2 together with the nitrogen atom to which they are attached form a pyrrolidino, piperidino or morpholino; Y Z is an amino acid sequence or derivative thereof, sufficient for substrate recognition and to which is optionally coupled a fluorescent donor moiety, a fluorescent acceptor moiety, an affinity tag or a detectable tag; or Z is an amino compound or aromatic phenoxy whose fluorescence or absorbance characteristics change after acylation with an amino acid; or Z is amino, alkylamino of 1 to 6 carbon atoms or lower alkoxy; with the condition of (1) when one of X and Z comprises a fluorescent donor, then the other of X and Z comprises a fluorescent acceptor radical, and (2) that when one of X and Z comprises an affinity tag then the other of X and Z understand a detectable marker; wherein the protease breaks or cleaves the substrate at the corresponding amide or ester link between Ala and Z.
2. The substrate according to claim 1, characterized in that Z is a phenoxy or amino aromatic radical derived by elimination of a hydrogen from the appropriate amino group or appropriate hydroxyl, respectively, of 7-amino-4-methylcoumarin; 6-amino-1-naphthalenesulfonamide; rhodamine 110; 2-naphthylamine; 4-methoxy-2-naphthylamine; 4-nor troaniline; or 4-nitrophenol.
3. The substrate according to claim 1, characterized in that it is covalently linked to a fluorescent donor moiety and to a fluorescent acceptor moiety, the donor and acceptor moieties are separated by the alanine residue and 7 to 12 additional amino acid residues.
4. The substrate according to claim 3, characterized in that the fluorescent donor moiety is selected from 2-aminobenzoyl and halogenated derivatives thereof; 5-. { (2-aminoethyl) amino} Naphthalene-1-sulfonyl; 5-dimethylaminonaphthalene-1-sulfonyl; 7-methoxycoumarin-4-acetyl; nicotinic acid and derivatives thereof; and tryptophan; and the fluorescent acceptor is selected from 3-nitrotyrosine; 4-nitrophenylalanine; 2,4-dinitrophenylalanine; 5- (dimethylamino) naphthalene-1-sulfonyl; 4- . { . { 4- (dimethylamino) phenyl} azo } benzoyl; or 4- (dimethylamino) azobenzene-4'-sulfonyl.
5. The substrate according to claim 3, characterized in that the donor / acceptor pairs are selected from the group consisting of: EDANS / DABCYL; tryptophan / 2,4-dinitrophenyl; tryptophan / dans i lo; 7-methoxycoumarin / 2,4-dinitrophenyl; 2-aminobenzoyl / 2,4-dinitrophenyl; and 2-aminobenzoyl / 3-nitrotyrosine.
6. The substrate according to claim 1, characterized in that the polypeptide is linked to a detectable label.
7. The substrate according to claim 6, characterized in that the detectable label is selected from a radioactive label or an indicator molecule such as β-galactosidase.
8. The substrate according to claim 1, characterized in that the substrate is linked to a detectable marker and an affinity tag.
9. The substrate according to claim 8, characterized in that the detectable label is selected from 125 I or β-galactosidase and the affinity tag is selected from biotin or a derivative thereof; a polyhistidine; a portion of amylose sugar; or a defined epitope, recognizable by a specific antibody.
10. The substrate according to claim 1, characterized in that the encapsulation group is selected from acetyl or succinimidyl.
11. The substrate according to claim 1, characterized in that it has the structure of ABz-Tbg-Tbg-Asn (Me) 2-Ala-Ser-Ser-Arg-Leu-Tyr (3-N02) -Ag-OH. [I KNOW THAT. ID. NO: 3].
12. The substrate according to claim 1, characterized in that it has the structure of N-acetyl-Tbg-Tbg-Asn (Me) 2-Ala-AMC [SEQ. ID. NO: 5]
13. The substrate according to claim 1, characterized in that it has the structure of d-biotin-Arg-Gly-Tbg-Tbg-Asn (Me) 2-Ala-Ser-Ser-Arg-Leu-Ala-Tyr-NH2 [SEQ. . ID. NO: 7].
14. The substrate according to claim 13, characterized in that the substrate is labeled with 125I.
15. The substrate according to claim 1, characterized in that it has the structure of Ac-Tbg-Tbg-Asn (Me) 2-Ala-pNP [SEQ. ID. NO: 8].
16. The substrate according to claim 1, characterized in that it has the structure of Ac-Tbg-Tbg-Asn (Me) 2-Ala-pNA [SEQ. ID. NO: 9].
17. A method for measuring the activity of a herpesvirus protease, characterized in that it comprises the steps of contacting the herpesvirus protease with a substrate according to claims 1, 11 or 12; and the periodic verification of the cleavage of said substrate.
18. The method according to claim 17, characterized in that the herpervirus is the cyto egalovirus.
19. The method according to claim 17, characterized in that the step of periodically verifying the cleavage of the substrate comprises the determination of a change in the size of said substrate.
20. The method according to claim 17, characterized in that the step of periodically verifying the cleavage of the substrate comprises the determination of a change in the fluorescence or absorbance of the substrate or the product.
21. The method according to claim 17, characterized in that the step of periodically verifying the cleavage of said substrate comprises the determination of a change in the solvent extraction capacity of the detectable label.
22. A method for identifying an inhibitor of a herpesvirus protease, characterized in that it comprises the steps of: (1) contacting the herpesvirus protease with a substrate as defined in claims 1, 11 or 12, in the presence of a test compound; and (2) periodically checking the cleavage of said substrate to determine the level of inhibition of protease activity, caused by the test compound.
23. The method according to claim 22, characterized in that the herpes virus is cytomegalovirus.
24. The method according to claim 22, characterized in that the step of periodically verifying the cleavage of the substrate comprises the determination of a change in the size of the substrate.
25. The method according to claim 22, characterized in that the step of periodically verifying the cleavage of the substrate comprises the determination of a change in the fluorescence or absorbance of the substrate or product.
26. The method according to claim 22, characterized in that the step of periodically verifying the cleavage of the substrate comprises the determination of a change in the solvent extraction capacity of the detectable label.
27. A device for measuring the activity of a herpesvirus protease, characterized in that it comprises: a herpesvirus protease, and a substrate as written according to claims 1, 11 or 12.
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US60/033,854 | 1996-12-27 |
Publications (1)
Publication Number | Publication Date |
---|---|
MXPA99006000A true MXPA99006000A (en) | 2001-05-17 |
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