| UPDATE | 09.99 |
| PATENT GRANT DATE | 14.09.99 |
| PATENT TITLE |
Reagent and kit for determining isoprenylating activity and inhibition thereof |
| PATENT ABSTRACT | The invention provides an immobilised oligopeptide for detecting protein prenylation consisting of an oligopeptide containing the amino acid sequence Xad-Xac-Xab-Xaa-OH at its carboxyl-terminus, at least one of Xaa, Xab, Xac and Xad representing cysteine (Cys), said sequence being capable of acting as a substrate for a prenyl transferase catalysing protein prenylation, and being bonded to a solid carrier, preferably at its amino-terminus. The invention further provides a kit for detecting protein prenylation comprising a first immobilised oligopeptide as mentioned above and a second immobilised oligopeptide which differs from said first immobilised oligopeptide in that the cysteine residue is substituted by another amino acid. Also provided are antibodies against the prenylated oligopeptides. |
| PATENT INVENTORS | Cohen; Louis Hartog (Voortwijk 10, NL-3621 HR Breukelen, NL); Nieuwenhuizen; Willem (Groeneweg 48, NL-3981 CL Bunnik, NL) |
| PATENT FILE DATE | 16.10.97 |
| PATENT FOREIGN APPLICATION PRIORITY DATA | Apr 20, 1993[EP] 93201146 |
| PATENT CLAIMS |
(a) An immobilized oligopeptide having 4-40 aa residues contg aa seq Xad-Xac-Xab-Xaa-OH at its carboxyl terminus, at least one of Xaa, Xab, Xac & Xad representing cysteine (Cys), said seq being capable of acting as substrate for prenyl transferase catalyzing protein prenylation, said oligopeptide bomded to solid carrier (b) MAb which binds reaction prodt of farnesyl pyrophosphate or geranylgeranyl pyrophosphate & said oligopeptide but not the oligopeptide |
| PATENT EXAMPLES |
EXAMPLE 1 (i) Synthesis of peptides to be used in the isoprenylation reaction Using 9-(fluorenylmethyl)oxycarbonyl (Fmoc) derivatives of the appropriate amino acids, peptides were synthesized by means of a semi-automatic solid-phase peptide synthesiser (Labortek, Bubendork Switzerland) using dicyclohexyl carbodiimide/8-hydroxybenzotriazole. The solid phase was 1% cross-linked 4-(hydroxymethyl)phenoxymethyl-polystyrene (Wang resin) charged with about 1 mmol/g of a Fmoc-methionine derivate. After each acylation step the Fmoc group of the peptide was cleaved with piperidine. Three peptides were synthesized, which had the following amino acid sequences: Peptide A (derived from the C-terminus of human pre-p21.sup.N-Ras) (SEQ ID NO: 1) ›NH.sub.2 !-Acp-Met-Gly-Leu-Pro-Cys-Val-Val-Met-›COOH! .vertline. S-tert-butyl Acp (=.epsilon.-aminocaproic acid) was introduced for providing a unique amino group suitable for conjugation to a support, and as an additional spacer. The thiol group of the cysteine residue was protected by a tert-butylthio group, which was removed after coupling to Sepharose. Peptide B (control peptide with Cys replaced by Ala) ›NH.sub.2 !-Acp-Met-Gly-Leu-Pro-Ala-Val-Val-Met-›COOH! (SEQ ID NO: 2) Peptide C (the same as peptide A, but with C-terminal methionine replaced by leucine as a substrate for geranylgeranylation (Kinsella, B. T., Erdman, R. A. and Maltese, W. M., Proc. Nat. Acad. Sci. 88 (1991) 8934-8938). (SEQ ID NO: 3) ›NH.sub.2 !-Acp-Met-Gly-Leu-Pro-Cys-Val-Val-Leu-›COOH! .vertline. S-tert-butyl (ii) Coupling of peptides to a solid phase Peptides A, B and C were coupled to activated CH-Sepharose 4B beads through the free NH.sub.2 group according to the procedure as described by the manufacturer (Pharmacia). 0.8-5 nmol of the peptides were coupled to 1 .mu.l of Sepharose gel. The material was stored in 0.5M NaCl-0.1M Tris/HCl (ph 8) -20% ethanol at 4.degree. C. Before use the tert-butylthio group was removed from peptides A and C by resuspending the Sepharose beads in 10 mM DTT (dithiothreitol). (iii) Quality control and quantification of coupled peptides The quantity of peptides coupled to the Sepharose beads was determined by C-terminal amino acid analysis. 10 .mu.l of washed peptide A-, peptide B-, peptide C-Sepharose (pepAsep, pepBsep, pepCsep, resp.) or beads without peptide (control) were resuspended in 200 .mu.l of 0.2M of N-ethyl-morpholinium acetate (pH 8.0) containing 1 mg/ml prewashed Carboxypeptidase A (Serva) and 1 mM phenylmethylsulfonyl fluoride (as serine protease inhibitor). Incubation was performed for 20 h at 25.degree. C. with continuous shaking. After centrifugation (1 min; 13,000 rpm) the supernatant, containing the C-terminal amino acids, was lyophilised. These amino acids were quantitated using an automated amino acid analyser. The minor quantities of amino acids present in the supernatant of the control incubation (from autodigestion of Carboxypeptidase A) were subtracted from the values obtained with the peptide-coupled Sepharoses. Only either valine and methionine (pepAsep and pepBsep) or valine and leucine (pepCsep) were detected and the molar ratio of valine to methionine/leucine was 2 to 1 in all cases. From the absolute values the quantity of pmol peptide bound to Sepharose was calculated, which was in the range of 0.8-5 .mu.mol of peptide/ml of Sepharose. EXAMPLE II Assay of protein:farnesyl transferase activity in rabbit reticulocyte lysate Commercially available rabbit reticulocyte lysate contains enzymes necessary for protein-isoprenylation, such as protein:farnesyl transferase activity (Vorburger, K., Kitten, G. T. and Nigg, E. G., EMBO J. 8 (1989) 4007-4013) and protein:geranylgeranyl transferase (Maltese, W. A. and Robishaw, J. D., J. Biol. Chem. 265 (1990) 18071-18074). Using pepAsep (example I) as substrate and pepBsep (example I) as control the assay of protein:farnesyl transferase activity was performed as follows: In 25 .mu.l of a mixture containing 5 .mu.l of pepAsep or pepBsep (both containing an equal quantity of peptide), 13 .mu.l rabbit reticulocyte lysate (promega), 0.5 mM MgCl.sub.2, 1 mM DTT, 50 mM Tris-HCl (pH 7.4) and ›.sup.3 H!-farnesyl pyrophosphate (.sup.3 H-FPP; concentration as indicated in the figures; specific radioactivity 15 Ci/mmol; ARC, USA) the incubation was performed at 37.degree. C. for 30 min with continuous shaking. The reaction was terminated by addition of 1 ml 2% SDS, the beads were spun down and washed 3 times with 2% SDS under shaking for 45 min at 50.degree. C. The radioactivity bound to the Sepharose was measured in a Packaged Tricarb liquid scintillation counter. Several characteristics of the peptide-farnesylation reaction, e.g. the dependency on peptide concentration (at constant quantity of Sepharose beads; FIG. 1A), farnesyl pyrophosphate concentration (FIG. 1B) and incubation time (FIG. 1C), are shown in FIG. 1. From the differences in the results obtained with pepAsep and pepBsep it is clear that the reaction is strongly dependent on the presence of the cysteine residue in peptide A. For the determination of the protein:farnesyl transferase activity the .sup.3 H-counts bound to pepBsep (background control) are subtracted from the counts bound to pepAsep. EXAMPLE III Assay of protein:geranylgeranyl transferase activity in rabbit reticulocyte lysate With pepCsep (example I) and ›.sup.3 H!-geranylgeranyl pyrophosphate (.sup.3 H-GGPP) as substrates and pepBsep (example I) as control protein:geranylgeranyl transferase activity was detected in reticulocyte lysate. In this example, illustrated in FIG. 2, the 25 .mu.l of incubation mixture contained: 5 .mu.l of pepBsep or pepCsep (containing the indicated quantity of peptide), 13 .mu.l rabbit reticulocyte lysate (Promega), 0.5 mM MgCl.sub.2, 1 mM DTT, 50 mM Tris-HCl (pH 7.4) and 0.4 .mu.M .sup.3 H-GGPP (specific radio-activity 15 Ci/mmol; ARC, USA) the incubation was performed at 37.degree. C. for 30 min with continuous shaking. The reaction was terminated by addition of 1 ml 2% SDS, the beads were spun down and washed three times with 2% SDS with shaking for 45 min at 50.degree. C. The radioactivity bound to the Sepharose was measured in a Packard Tricarb liquid scintillation counter. The dependency of the peptide C concentration is shown in FIG. 2. Only a few dpm were associated with the control pepBsep. EXAMPLE IV Test of inhibitors of protein:farnesyl transferase activity in rabbit reticulocyte lysate In this reticulocyte lysate system two farnesyl pyrophosphate analogues (FPPAs), (E,E)-›Hydroxy-(3,7,11-trimethyl-2,6,10-dodecatrienyloxymethylphosphinyl)m ethyl!phosphonic acid (FPPA1) (Biller S. A. et al., J. Am. Chem. Soc. 133 (1991) 8522-8524) and farnesyl phosphonophosphate (FPPA2) (Valentijn, A.R.P.M., Van der Marel, G. A., Cohen, L. H, and Van Boom, J. H., SYNLETT (1991) 664--664) were tested for their potency to inhibit the protein:farnesyltransferase activity. The reaction was performed as described above, using 80 pmol of both peptides/6 .mu.l beads and 0.7 .mu.M .sup.3 H-FPP, in the presence of various concentrations of FPPA1 or FPPA2. As can be seen in FIG. 3, both analogues are inhibitors of the enzyme reaction. FPPA2 is more potent (IC.sub.50 -value of 0.29.+-.0.02 .mu.M) than FPPA1 (IC.sub.50 :5.26.+-.1.10 .mu.M) |
| PATENT PHOTOCOPY | Available on request |
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