Unnatural substrates of prenylation enzymens and antibodies that recognize unique moieties of prenylated proteins, which unique moieties are transferred from the unnatural substrates are used for detecting and isolating prenylated proteins, and for screening for inhibitors of prenylation enzymes

Andres, Douglas A.

Spielmann, H. Peter

English

University of Kentucky

University of KentuckyUKnowledgeMolecular and Cellular Biochemistry FacultyPatents Molecular and Cellular Biochemistry6-29-2010Antibodies and Unnatural Substrates of PrenylationEnzymes for Use in Detecting and IsolatingPrenylated ProteinsH. Peter SpielmannUniversity of Kentucky, hps@uky.eduDouglas A. AndresUniversity of Kentucky, dandres@uky.eduRight click to open a feedback form in a new tab to let us know how this document benefits you.Follow this and additional works at: https://uknowledge.uky.edu/biochem_patentsPart of the Medical Biochemistry CommonsThis Patent is brought to you for free and open access by the Molecular and Cellular Biochemistry at UKnowledge. It has been accepted for inclusion inMolecular and Cellular Biochemistry Faculty Patents by an authorized administrator of UKnowledge. For more information, please contactUKnowledge@lsv.uky.edu.Recommended CitationSpielmann, H. Peter and Andres, Douglas A., "Antibodies and Unnatural Substrates of Prenylation Enzymes for Use in Detecting andIsolating Prenylated Proteins" (2010). Molecular and Cellular Biochemistry Faculty Patents. 7.https://uknowledge.uky.edu/biochem_patents/7(12) United States Patent Spielmann et a1. US007745589B1 US 7,745,589 B1 Jun. 29, 2010 (10) Patent N0.: (45) Date of Patent: (54) ANTIBODIES AND UNNATURAL SUBSTRATES OF PRENYLATION ENZYMES FOR USE IN DETECTING AND ISOLATING PRENYLATED PROTEINS (75) Inventors: Hans Peter Spielmann, Lexington, KY (US); Douglas A. Andres, Lexington, KY (Us) (73) Assignee: University of Kentucky Research Foundation, Lexington, KY (U S) ( * ) Notice: Subject to any disclaimer, the term of this patent is extended or adjusted under 35 U.S.C. 154(b) by 845 days. (21) App1.No.: 11/459,299 (22) Filed: Jul. 21, 2006 Related US. Application Data (60) Provisional application No. 60/701,162, ?led on Jul. 21,2005. (51) Int. Cl. C07K 16/00 (2006.01) (52) us. c1. ............. .. 530/389.8;530/388.9;424/175.1 (58) Field of Classi?cation Search ......... .. None See application ?le for complete search hlstory. (56) References Cited OTHER PUBLICATIONS Campbell, A (Laboratory Techniques in Biochemistry and Molecular Biology, vol. 13, Chapter 1, pp. 1-33, 1984).* Andres, D. A., Crick, D. C., Finlin, B. S., and Waechter, C. J. (1999) Rapid identi?cation of cysteine-linked isoprenyl groups by meta bolic labeling with [3H]farnesol and [3H]-geranylgeraniol. Methods Mol. Biol. 116, 107-23. Baron, R., Fourcade, E., Lajoie-Mazenc, I., Allal, C., Couderc, B., Barbaras, R., Favre, G., Faye, J. C., and Pradines, A. (2000) RhoB prenylation is driven by the three carboxyl-terminal amino acids of the protein: evidence in vivo by an anti-farnesyl cysteine antibody. Proc. Natl. Acad. Sci. USA. 97, 11626-31. Berndt, P., Fields, G. B., and Tirrell, M. (1995) Synthetic Lipidation of Peptides and Amino Acids: Monolayer Structure and Properties. J. Am. Chem. Soc. 117, 9515-9522. Chehade, K. A., Andres, D. A., Morimoto, H., and Spielmann, H. P. (2000) design and synthesis of a transferable farnesyl pyrophosphate analogue to Ras by protein farnesyltransferase. J. Org. Chem. 65, 3027-33. Chehade, K. A., Kiegiel, K., Isaacs, R. J., Pickett, J. S., Bowers, K. E., Fierke, C. A., Andres, D. A., and Spielmann, H. P. (2002) Photoaf ?nity analogues of farnesyl pyrophosphate transferable by protein farnesyl transferase. J. Am. Chem. Soc. 124, 8206-19. Chehade, K. A. H., Andres, D. A., Morimoto, H., and Spielmann, H. P. (2000) Design and Synthesis of a Transferable Farnesyl Pyrophosphate. J. Org. Chem. 65, 3207-3033. Crick, D. C., et al., (1998) Geranyl geraniol overcomes the block of cell proliferation by lovastatin in C6 glioma cells. J. Med. Chem. 38(8), 1372-9. Crick, D. C., Andres, D. A., andWaechter, C. J. (1997) Novel salvage pathway utilizing farnesol and geranylgeraniol for protein isoprenylation. Biochem. Biophys. Res. Commun. 237, 483-7. Crick, D. C., Andres, D. A., and Waechter, C. J. (1995) Farnesol is utilized for protein isoprenylation and the biosynthesis of cholesterol in mammalian cells. Biochem. Biophys. Res. Commun. 211, 590-9. Danesi, R., McLellan, C. A., and Myers, C. E., (1995) Speci?c Labeling of Isoprenylated Proteins: Application to Study Inhibitors of the Post-Translational Farnesylation and Geranylgeraniolation. Biochm. Biophys. Res. Comm., 206(2), 637-43. Dechat, Thomas, Takeshi Shimi, Stephen A. Adam, Antonio E. Rusinol, Douglas A. Andres, H. Peter Spielmann, Michael S. Sinensky, and Robert D. Goldman, (2007) Alterations in Mitosis and Cell Cycle Progression Caused by a Mutant Lamin A Known to Accelerate Human Aging. PNAS 104, 12, 4455-4960. Dursina, B., et al., (2006) Identi?cation and speci?city pro?ling of protein prenyltransferase inhibitors using new ?uorescent phosphoisoprenoids. J. Am. Chem. Soc. 128(9), 2822-35. Eummer, J. T., et a1. (1999) Novel Limonene Phosphate and Farnesyl Dipho sphate Analogues: Design, Synthesis, and Evaluation as Poten tial Protein-Farnesyl Transferase Inhibitors. Biorg. and Med Chem., 7, 241-50. Gibbs, B. S., Zahn, T. J., Mu,Y., Sebolt-Leopold, J. S., and Gibbs, R. A. (1999) Novel Farnesol and Geranylgeraniol Analogues: A Poten tial New Class of Anticancer Agents Directed against Protein Prenylation. J. Med. Chem. 42, 3800-3808. Holstein, S. A., Wohlford-Lenane, C. L., Wiemer, D. F., and Hohl, R. J. (2003) Isoprenoid Pyrophosphate Analogues Regulate Expression of Ras-Related Proteins. Biochemistry 42, 4384-4391. Kho, Y., Kim, S. C., Jiang, C., Barma, D., Kwon, S. W., Cheng, J., Jaunbergs, J., Weinbaum, C., Tamanoi, F., Falck, J., and Zhao, Y. (2004) A tagging-via-substrate technology for detection and proteomics of farnesylated proteins. Proc. Natl. Acad. Sci. USA. 101,12479-84. Kim, K.W., et al., (2001) Inactivation of farnesyltransferase and geranylgeranyl-transferase I by caspase-3: cleavage of the common alpha subunit during apoptosis. Oncogene. 20(3), 358-66. Lin, H. P., Hsu, S. C., Wu, J. C., Sheen, I. J.,Yan, B. S., and Syu, W. J. (1999) Localization of isoprenylated antigen of hepatitis deltavirus by anti-farnesyl antibodies. J. Gen. Virol. 80 (Pt 1), 91-6. Liu, X. H., Suh, D.Y., Call, J., and Prestwich, G. D. (2004) Antigenic prenylated peptide conjugates and polyclonal antibodies to detect protein prenylation. Bioconjugate Chem. 15, 270-7. Quellhorst, G. J., C.M. Allen, and M. Wessling-Resnick, (2001) Modi?cation of Rab5 with a photoactivatable analog of geranylgeranyl diphosphate. J Biol Chem. 276(44), 40727-40733. Rowell, C. A., Kowalczyk, J. J., Lewis, M. D., and Garcia, A. M. (1997) Direct demostration of geranylgeranylation and farnesylation of Ki-Ras in vivo, J. Biol. Chem. 272, 14093-7. Thai, L., Rush, J. S., Maul, J. E., Devarenne, T., Rodgers, D. L., Chappell, J., and Waechter, C. J. (1999) Farnesol is utilized for isoprenoid biosynthesis in plant cells via farnesyl pyrophosphate formed by successive monophosphorylation reactions. Proc. Natl. Acad. Sci. USA. 96, 13080-5. Troutman J. M., Chehade, K. A., Kiegiel, K., Andres, D.A., Speilmann, H. P. (2004) Synthesis of acyloxyrnethyl ester prodrugs of the transferable protein farnesyl transferase substrate farnesyl methylenediphosphonate. Bioorg Med chem. Lett. 14 (19), 4979-82. * cited by examiner Primary ExamineriSha?qul Haq (74) Attorney, Agent, or FirmiStites & Harbison PLLC; Mandy Wilson Decker (57) ABSTRACT Unnatural substrates of prenylation enzymens and antibodies that recognize unique moieties of prenylated proteins, which unique moieties are transferred from the unnatural substrates are used for detecting and isolating prenylated proteins, and for screening for inhibitors of prenylation enzymes. 10 Claims, 20 Drawing Sheets US. Patent Jun. 29, 2010 Sheet 8 0f 20 I00 Provide a prenylatinn reactian group including: a. prenyl all on enzyme, an unnatural sabstrate to the px‘anylation enzyme having a unique mcaiety 01' av precursor to the unnatural su'hsimm, and an antibody that recognizes the unique moieiy 102 Provide a sample containing at least une protein Introduce the sample ‘t0 the prenylatien enzymra, and the unnatural substrate 0:‘ precursor [0 the unnatural subsn'ate Y l 06 US 7,745,589 B1 104 Detect resulting prenylaled pmte'ins using the aniilmzly m“ the group [/1 l'solate resulting preny'lated proteins using the antibody 0F the gmup \ 108 H 0 FIG URE- 6 US. Patent 206 Z30 Jun. 29, 2010 Sheet 9 0f 20 PYDVidE a prenylatinn macticm gmup including: a prenyiatian enzyme, an unnamra! substrate in he prenylaliun enzyme hmiing a unique moieiy m" a precursor m the unnatumi suhslmte, and an an‘iibndy {ha} recognizes the 'unklue momty ‘F Prepare a sample including: at least one protein capable Qf'being many ! axed, ?u: prenymtion enzyme, the unnatural substrate, and a‘ candidate prenyiaiinn enzyme inhibitor Allow contan‘is Q'Hhe sample '10 interact 1? Detect the remaking, prenyhtcd pmieins in the sample using the antibady R F Idantify ths candidate pmnylaticm enzyme inhibitm' as an actual} prenyimian anzyime inhibitor when {he dammed prenylated proteins ara belnw a predetermined level FIGU RE YA US 7,745,589 B1 204 208 US. Patent 304 308 Jun. 29, 2010 Sheet 10 0f 20 Prtwide a prenyiatinn reavctien group inciutiing: a prenyiatimt magma, an unnaturai substrate to the prcnyiatiun enzyme IRS-‘113g a unique in?ict}: at a pres-timer t0 the unnaturai substrate, and an antibcid'y that rccagnizas the unique mulcty US 7,745,589 B1 300 Prcpart: a ?rst sampie somaining at least one protein capable of being prcnyiated, the prenyiatinn enzyme, the unnatural substrate, and a candidate pretty I at] on enzyme inhibitur Prepare a secund sample containing at least one protein capable at‘ being prenylated, this prenft‘iation enzyme, and the unnatutal substrate K 306 Ailnw the contents of the ?rst sampl a to interact Allow the mutants of the second sam'pie to interact \ v 31.0 Detect the resuiting pmnylated proteins in the ?rst sample using the antibody Detect that ttzsuiting, prenylated pmteins in the second sampie using, the antibody V sac-0nd sam'pie Compare the detected prenylated proteins in time ?rst sampie with the datactcd prenylatmt proteins in the I identify the: candidate prenytation enzyme: inhibitor as an actual prenyiatiun enzyme inhibitor when the detected prenyiaited pi'meins in the ?rst sampie is less than the detected pr?nyiated proteins in the second sample FIGURE 73 316 US. Patent Jun. 29, 2010 Sheet 11 0f 20 AG-Ab EUSA Ab Diiution FIG “RE SA D 1D C) 4:- m m Ab Dilution FIGURE 83 US 7,745,589 B1 US. Patent Jun. 29, 2010 Sheet 12 0f 20 US 7,745,589 B1 intensity Q Q Q Q 1:“ E" in a“ c: c: c: :2 Ab Dilutinn FIG [E R E SC US. Patent Jun. 29, 2010 Sheet 13 0f 20 US 7,745,589 B1 0 NH; 0 0 Q Q {3H Rum” N N H H 0 0 5R1 M NH NW (Herminal pm'tion ofmodi?ad RD]? Protein R, m H R; famesy] IR; E-aniiinogermwi R] : pNOg?aniiinogemnyi FIG {.3 RE 95% OH is o R‘ ‘:3 OH w-Raa’f N ‘ N ' H H G 0 SR; {Ti-terminal po?ian nfmocii?ed ERAS Protein R\ H R, famesyl R; m ‘3amiinugeranyi R}, p?ikmniiinngeranyi FIGURE 93 U S. Patent Jun. 29, 2010 Sheet 14 0f 20 US 7,745,589 B1 F 1G URE 10B ENSURE '1 0(‘ “(HERE 10D 1US. Patent Jun. 29, 2010 Sheet 15 0f 20 US 7,745,589 B1 wwméizua wgmééus gméqae m.éméqé wwwwig mmxéib, G A n a . . “an FIGURE 11 A 3%32 Ex mmméqéum wwméia G A N L. m a EmiiMA :3 FIGURE 11B US. Patent Jun. 29, 2010 Sheet 16 0f 20 US 7,745,589 B1 97km n.- ~ 66km _. i?kD? --+ 31 kDa my» 212K516 up» MkDa up FIG-U RE 12 US. Patent Jun. 29, 2010 Sheet 17 0f 20 US 7,745,589 B1 b “w G A ,_ cm. hang»,_ cm mn?m3333533m .6 Pmmmb,5 aFIGURE 13 US. Patent Jun. 29, 2010 Sheet 18 0f 20 US 7,745,589 B1 Q7 kDa ,,,,,,,,,, ‘ »» Da-w *- AGJabeisd preteins QAERDa-Q» FQGURE 15 US. Patent Jun. 29, 2010 Sheet 19 0f 20 US 7,745,589 B1 ....................................... . . aim“ B. o a_ mama .mmmmmE $3-? . g “ . U mama mé %mama, , I?@5555, ............... gnaw 5 1, 842nu.HumwEQEQw Sum 

Antibodies and Unnatural Substrates of Prenylation Enzymes for Use in Detecting and Isolating Prenylated Proteins

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Antibodies and Unnatural Substrates of Prenylation Enzymes for Use in Detecting and Isolating Prenylated Proteins

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