- Email: [email protected]
Generation of a novel anti-geldanamycin antibody
BBRC Biochemical and Biophysical Research Communications 330 (2005) 561–564 www.elsevier.com/locate/ybbrc
Generation of a novel anti-geldanamycin antibody Eran Barzilay a, Nathalie Ben-Califa a, Mika Shahar a, Yoel Kashman b, Drorit Neumann a,* a
Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel-Aviv University, Ramat-Aviv 69978, Israel b School of Chemistry, Tel-Aviv University, Ramat-Aviv 69978, Israel Received 2 March 2005 Available online 18 March 2005
Abstract Geldanamycin (GA) and herbimycin A are benzoquinone ansamycins (BAs) that inhibit the molecular chaperone HSP90. The central role of HSP90 in maintaining the conformation, stability, and function of key oncogenic proteins involved in signal transduction pathways renders BAs attractive candidates for clinical development. Two GA derivatives, 17-allylamino-17-demethoxygeldanamycin and 17-demethoxy-17-N,N-dimethylaminoethylamino-geldanamycin are currently evaluated in clinical trials. The present study demonstrates generation of a polyclonal antibody elicited against GA that was conjugated to keyhole limpet hemocyanin via its 17 position. The anti-GA antibody recognizes GA as well as other BAs, suggesting its possible application for monitoring plasma levels of GA derivatives. The specificity of the antibody towards BAs is demonstrated by its inability to recognize radicicol, an HSP90 inhibitor not related to BAs. This antibody thus presents a novel research tool as well as a possible alternative approach for monitoring drug levels in patients. Ó 2005 Elsevier Inc. All rights reserved. Keywords: Geldanamycin; Herbimycin A; 17-AAG; 17-DMAG; Antibody; Benzoquinone ansamycins; HSP90
Benzoquinone ansamycins (BAs), such as geldanamycin (GA) and herbimycin A (HA), are a family of antibiotics that specifically inhibit the cytosolic chaperone HSP90 and its endoplasmic reticulum homologue GRP94 [1,2]. Inhibition of HSP90 promotes the degradation of HSP90 client proteins. Among these are Tyr kinases [3] such as epidermal growth factor receptor [4], p185erb-B2 [5,6], and viral Src [7], as well as other proteins such as the cystic fibrosis transmembrane conductance regulator (CFTR) protein [8], mutated p53 [9], Raf-1 [10], CDK4 [11], and steroid hormone receptors [12]. ATP binding to HSP90 was proposed to regulate the interaction of HSP90 with co-chaperones [13,14]. GA binds to the ATP binding pocket of HSP90, thus preventing ATP binding to this site [15,16]. Since many HSP90 *
Corresponding author. Fax: +972 3 6407432. E-mail address: [email protected] (D. Neumann).
0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.03.014
client proteins are involved in cell proliferation and malignant transformation, HSP90 inhibitors have clinical potential in cancer management. Two GA derivatives, namely, 17-allylamino-17-demethoxygeldanamycin (17-AAG) and 17-demethoxy-17-N,N-dimethylaminoethylamino-geldanamycin (17-DMAG), are currently implemented in clinical trials for the treatment of cancer [17–19]. This communication describes the production and characterization of a GA-specific antibody, which also recognizes other BAs such as HA, 17-AAG, and 17-DMAG.
Materials and methods Materials. Radicicol, herbimycin A, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), and glutaraldehyde were obtained from Sigma (St. Louis, MO), while Affi-Gel 10 resin was obtained from Bio-Rad (Hercules, CA), GA was kindly provided by
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E. Barzilay et al. / Biochemical and Biophysical Research Communications 330 (2005) 561–564
D. Newman of the National Cancer Institute (Rockville, MD). 17AAG and 17-DMAG were obtained from Kosan Biosciences (Hayward, CA). 17-Hexamethylenediamine derivatization of GA. 17-Hexamethylenediamine derivatization was performed essentially as described [7] to generate a GA derivative that can be covalently immobilized via its amino moiety. GA (10 mM in CHCl3) was reacted with 1,6-hexanediamine at 1:10 molar ratio, respectively, for 2 h in the dark, to generate quantitatively a single derivative, the required 17-hexamethylenediamine-17-demethoxygeldanamycin (HDG-GA, Fig. 1). HDG-GA was dissolved in DMSO to obtain a stock solution of 10 mg/ml. The chemical structure of the product was verified by NMR analysis. NMR (500 MHz, CDCl3) 9.20brs(NH), 7.28 s, 6.95d (J = 11 Hz), 6.58t (11 Hz), 6.25 m, 5.86 m (2H), 5.19 s, 4.80 m (NH2) 4.31d (J = 10), 3.57 m, 3.45 m, 3.63 s (OMe), 3.27 s (OMe), 3.02 m, 2.66 m (2H), 2.30 m, 2.04 s (CH3), 1.79 s (CH3), and 1.70 m. Conjugation of HDG-GA. Carrier proteins (KLH or BSA) were dissolved in double-distilled water (DDW) to a concentration of 10 mg/ml. The protein solution was mixed with HDG-GA at a volume ratio of 2:1, respectively. The mixture was titered to a pH of 8.5 with NaOH. Glutaraldehyde (1% in DDW) was added to the GA-carrier protein mixture at a volume ratio of 2:3, respectively. The mixture was incubated at room temperature in the dark for 24 h, followed by dialysis against phosphate-buffered saline (PBS), pH 7.4. Immobilization of HDG-GA on Affi-Gel 10 resin (Bio-Rad) was performed according to the manufacturerÕs instructions. Affi-Gel beads were washed with 5 bed volumes of cold isopropanol. Beads were then incubated with HDG-GA (1 bed volume, 5 mg/ml in DMSO) for 4 h in the dark at room temperature, followed by blocking in 100 mM ethanolamine (pH 7.5). Antibody production. Anti-GA antiserum was obtained by immunization of rabbits with the KLH-GA conjugate. For the first injection, 0.5 mg of KLH-GA conjugate (calculated on the basis of KLH content) in 0.5 ml PBS was emulsified (1:1) with complete FreundÕs adjuvant (Difco, Detroit, MI) and injected subcutaneously. Four booster injections of 0.5 mg KLH-GA conjugate emulsified with incomplete FreundÕs adjuvant were performed at three-week intervals.
The serum was subjected to affinity purification using the immobilized HDG-GA beads. Western blots and immunoblot inhibition assays. Western blotting was performed essentially as described [20]. For immunoblot inhibition assays, BSA-GA conjugate (1 lg/lane) was separated on 7.5% SDS– PAGE and transferred to a nitrocellulose membrane. The anti-GA antiserum (1:1000 in 5% skim milk) was incubated for 1 h at room temperature with GA (5 lM), prior to incubation with the nitrocellulose membrane. ELISA. Ninety-six-well plates were coated with 0.2 lg/well BSAGA. The affinity purified anti-GA antibody (50 ng/ml in 5% skim milk) was incubated with the inhibitory drug for 1 h at room temperature and was subsequently used for probing the BSA-GA-coated wells. Wells were then incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody. Detection was performed at 450 nm.
Results and discussion Generation of an anti-GA antibody To generate an antibody that would specifically recognize GA and its 17 position derivatives (Fig. 1, R1), we conjugated HDG-GA via its 17 position to the carrier protein. We rationalized that this mode of conjugation would render the remaining sites of GA accessible to antibody binding and thus allow the antibody to recognize GA and its 17-position derivatives, 17-AAG and 17-DMAG. The anti-GA antiserum was obtained by immunization of rabbits with the KLH-GA conjugate and was subjected to affinity purification using the immobilized HDG-GA beads. The anti-GA antibody recognizes both conjugated and free GA
Fig. 1. Schematic structure of benzoquinone ansamycins employed in this study. Geldanamycin (GA), herbimycin A (HA), 17-allylamino17-demethoxygeldanamycin (17-AAG), 17-demethoxy-17-N,N-dimethylaminoethylamino-geldanamycin (17-DMAG), and 17-hexamethylenediamine-17-demethoxygeldanamycin (HDG-GA).
To assess whether the antibody recognizes GA, we employed the BSA-conjugated GA. The BSA-GA conjugate was separated on 7.5% SDS–PAGE and transferred to a nitrocellulose membrane. BSA that was subjected to the same conditions as for conjugation was used as a control. Subsequently, the membrane was probed with the crude anti-GA antiserum (1:1000 in 5% skim milk). The results depicted in Fig. 2 (left panel) demonstrate that the antiserum specifically recognizes the BSA-GA conjugate. Next, we questioned whether the antiserum could recognize free, unconjugated GA. To address this, the anti-GA antiserum was incubated for 1 h at room temperature in the presence of GA (5 lM), prior to incubation with BSA-GA or mock-conjugated BSA blotted onto a nitrocellulose membrane. Pre-incubation of the anti-GA antiserum with free GA reduced antibody binding to background levels (Fig. 2, middle panel), thus demonstrating that the anti-GA antiserum recognizes free GA. GA-recognition by the affinity purified anti-GA antibody was also demonstrated by its reactivity with BSA-GA in Western blot analysis (Fig. 2, right panel).
E. Barzilay et al. / Biochemical and Biophysical Research Communications 330 (2005) 561–564
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Fig. 3. Recognition of different BAs by the anti-GA antibody. The anti-GA antibody (50 ng/ml in 5% skim milk) was incubated with the inhibitory drug for 1 h at room temperature and was subsequently used for probing ELISA plates coated with the BSA-GA conjugate (0.2 lg/well). The data are represented as percent of control-uninhibited binding of the anti-GA antibody to BSA-GA. Fig. 2. Binding of the anti-GA antibody to BSA-GA. GA conjugated to BSA and mock-conjugated BSA (1 lg/lane) were separated on 7.5% SDS–PAGE and transferred onto nitrocellulose membrane filters. The membrane filters were subsequently probed with the anti-GA antiserum (1:1000, left panel), anti-GA antiserum that was pre-incubated with GA (5 lM, middle panel) or the affinity purified anti-GA antibody (50 ng/ml, right panel). The BSA-GA conjugate (solid arrowhead) is accompanied by BSA-GA dimers (empty arrowhead) and high-order complexes, due to the glutaraldehyde-conjugation process. A similar pattern is observed in the mock-conjugated BSA.
[17–19] emphasizes the importance of the anti-GA antibody we have produced as it, or refined versions of it, may now be considered as powerful tools for monitoring the levels of the drug in the plasma of BA treated patients and for assessment of pharmacokinetics and pharmacodynamics of these drugs. Availability of the antibodies would thus be of particular importance for furthering the ongoing clinical studies.
Acknowledgments The anti-GA antibody recognizes other benzoquinone ansamycins We then raised the question of whether the anti-GA antibody recognizes BAs other than GA. Towards this end, we used the ELISA technique to determine the capacity of various BAs to inhibit binding of the antiGA antibody to BSA-GA-coated plates. The inhibitory compounds (GA, HA, 17-AAG or 17-DMAG) at concentrations ranging from 0.03 to 30 lM were added to the affinity purified anti-GA antibody (50 ng/ml in 5% skim milk) prior to its addition to the BSA-GA-coated wells. As a control we employed radicicol, a non-BA HSP90 inhibitor. Fig. 3 demonstrates that while radicicol did not interfere with antibody binding to the plates, all the BAs used in this assay inhibited antibody binding in a dose dependent manner. Moreover, all BAs examined were equally potent in inhibiting antibody binding to the BSA-GA-coated plates. The fact that HA was as potent as GA and as its 17-position derivatives in inhibiting binding of antibody to immobilized GA (Fig. 3) suggests that not all positions in the GA molecule take part in the antigenic determinants recognized by the anti-GA antibody. The antibody thus has a significant potential for further research on these drugs. Moreover, introduction of 17-AAG and 17-DMAG to clinical trials
This work was supported by a grant from the Israel Science Foundation administered by the Israel Academy of Sciences and Humanities (574/99 18.2) to D.N. This work was carried out in partial fulfillment of the requirements for the Ph.D. degree of Eran Barzilay from the Sackler Faculty of Medicine, Tel-Aviv University, Israel.
References [1] W.B. Pratt, The hsp90-based chaperone system: involvement in signal transduction from a variety of hormone and growth factor receptors, Proc. Soc. Exp. Biol. Med. 217 (1998) 420–434. [2] L. Neckers, T.W. Schulte, E. Mimnaugh, Geldanamycin as a potential anti-cancer agent: its molecular target and biochemical activity, Invest. New Drugs 17 (1999) 361–373. [3] L. Sepp-Lorenzino, Z. Ma, D.E. Lebwohl, A. Vinitsky, N. Rosen, Herbimycin A induces the 20S proteasome- and ubiquitindependent degradation of receptor tyrosine kinases, J. Biol. Chem. 270 (1995) 16580–16587. [4] M. Sakagami, P. Morrison, W.J. Welch, Benzoquinoid ansamycins (herbimycin A and geldanamycin) interfere with the maturation of growth factor receptor tyrosine kinases, Cell Stress Chaperones 4 (1999) 19–28. [5] P. Miller, C. DiOrio, M. Moyer, R.C. Schnur, A. Bruskin, W. Cullen, J.D. Moyer, Depletion of the erbB-2 gene product p185 by benzoquinoid ansamycins, Cancer Res. 54 (1994) 2724–2730.
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[6] W. Xu, E. Mimnaugh, M.F. Rosser, C. Nicchitta, M. Marcu, Y. Yarden, L. Neckers, Sensitivity of mature Erbb2 to geldanamycin is conferred by its kinase domain and is mediated by the chaperone protein Hsp90, J. Biol. Chem. 276 (2001) 3702–3708. [7] L. Whitesell, E.G. Mimnaugh, B. De Costa, C.E. Myers, L.M. Neckers, Inhibition of heat shock protein HSP90-pp60v-src heteroprotein complex formation by benzoquinone ansamycins: essential role for stress proteins in oncogenic transformation, Proc. Natl. Acad. Sci. USA 91 (1994) 8324–8328. [8] M.A. Loo, T.J. Jensen, L. Cui, Y. Hou, X.B. Chang, J.R. Riordan, Perturbation of Hsp90 interaction with nascent CFTR prevents its maturation and accelerates its degradation by the proteasome, EMBO J. 17 (1998) 6879–6887. [9] M.V. Blagosklonny, J. Toretsky, L. Neckers, Geldanamycin selectively destabilizes and conformationally alters mutated p53, Oncogene 11 (1995) 933–939. [10] T.W. Schulte, M.V. Blagosklonny, C. Ingui, L. Neckers, Disruption of the Raf-1-Hsp90 molecular complex results in destabilization of Raf-1 and loss of Raf-1-Ras association, J. Biol. Chem. 270 (1995) 24585–24588. [11] L. Stepanova, X. Leng, S.B. Parker, J.W. Harper, Mammalian p50Cdc37 is a protein kinase-targeting subunit of Hsp90 that binds and stabilizes Cdk4, Genes Dev. 10 (1996) 1491–1502. [12] L. Whitesell, P. Cook, Stable and specific binding of heat shock protein 90 by geldanamycin disrupts glucocorticoid receptor function in intact cells, Mol. Endocrinol. 10 (1996) 705–712. [13] J. Johnson, R. Corbisier, B. Stensgard, D. Toft, The involvement of p23, hsp90, and immunophilins in the assembly of progesterone
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receptor complexes, J. Steroid Biochem. Mol. Biol. 56 (1996) 31– 37. W. Sullivan, B. Stensgard, G. Caucutt, B. Bartha, N. McMahon, E.S. Alnemri, G. Litwack, D. Toft, Nucleotides and two functional states of hsp90, J. Biol. Chem. 272 (1997) 8007–8012. C. Prodromou, S.M. Roe, R. OÕBrien, J.E. Ladbury, P.W. Piper, L.H. Pearl, Identification and structural characterization of the ATP/ADP-binding site in the Hsp90 molecular chaperone, Cell 90 (1997) 65–75. S.M. Roe, C. Prodromou, R. OÕBrien, J.E. Ladbury, P.W. Piper, L.H. Pearl, Structural basis for inhibition of the Hsp90 molecular chaperone by the antitumor antibiotics radicicol and geldanamycin, J. Med. Chem. 42 (1999) 260–266. E.A. Sausville, J.E. Tomaszewski, P. Ivy, Clinical development of 17-allylamino, 17-demethoxygeldanamycin, Curr. Cancer Drug Targets 3 (2003) 377–383. T.W. Schulte, L.M. Neckers, The benzoquinone ansamycin 17allylamino-17-demethoxygeldanamycin binds to HSP90 and shares important biologic activities with geldanamycin, Cancer Chemother. Pharmacol. 42 (1998) 273–279. Z.Q. Tian, Y. Liu, D. Zhang, Z. Wang, S.D. Dong, C.W. Carreras, Y. Zhou, G. Rastelli, D.V. Santi, D.C. Myles, Synthesis and biological activities of novel 17-aminogeldanamycin derivatives, Bioorg. Med. Chem. 12 (2004) 5317–5329. D. Neumann, L. Wikstrom, S.S. Watowich, H.F. Lodish, Intermediates in degradation of the erythropoietin receptor accumulate and are degraded in lysosomes, J. Biol. Chem. 268 (1993) 13639– 13649.
Generation of a novel anti-geldanamycin antibody Eran Barzilay a, Nathalie Ben-Califa a, Mika Shahar a, Yoel Kashman b, Drorit Neumann a,* a
Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel-Aviv University, Ramat-Aviv 69978, Israel b School of Chemistry, Tel-Aviv University, Ramat-Aviv 69978, Israel Received 2 March 2005 Available online 18 March 2005
Abstract Geldanamycin (GA) and herbimycin A are benzoquinone ansamycins (BAs) that inhibit the molecular chaperone HSP90. The central role of HSP90 in maintaining the conformation, stability, and function of key oncogenic proteins involved in signal transduction pathways renders BAs attractive candidates for clinical development. Two GA derivatives, 17-allylamino-17-demethoxygeldanamycin and 17-demethoxy-17-N,N-dimethylaminoethylamino-geldanamycin are currently evaluated in clinical trials. The present study demonstrates generation of a polyclonal antibody elicited against GA that was conjugated to keyhole limpet hemocyanin via its 17 position. The anti-GA antibody recognizes GA as well as other BAs, suggesting its possible application for monitoring plasma levels of GA derivatives. The specificity of the antibody towards BAs is demonstrated by its inability to recognize radicicol, an HSP90 inhibitor not related to BAs. This antibody thus presents a novel research tool as well as a possible alternative approach for monitoring drug levels in patients. Ó 2005 Elsevier Inc. All rights reserved. Keywords: Geldanamycin; Herbimycin A; 17-AAG; 17-DMAG; Antibody; Benzoquinone ansamycins; HSP90
Benzoquinone ansamycins (BAs), such as geldanamycin (GA) and herbimycin A (HA), are a family of antibiotics that specifically inhibit the cytosolic chaperone HSP90 and its endoplasmic reticulum homologue GRP94 [1,2]. Inhibition of HSP90 promotes the degradation of HSP90 client proteins. Among these are Tyr kinases [3] such as epidermal growth factor receptor [4], p185erb-B2 [5,6], and viral Src [7], as well as other proteins such as the cystic fibrosis transmembrane conductance regulator (CFTR) protein [8], mutated p53 [9], Raf-1 [10], CDK4 [11], and steroid hormone receptors [12]. ATP binding to HSP90 was proposed to regulate the interaction of HSP90 with co-chaperones [13,14]. GA binds to the ATP binding pocket of HSP90, thus preventing ATP binding to this site [15,16]. Since many HSP90 *
Corresponding author. Fax: +972 3 6407432. E-mail address: [email protected] (D. Neumann).
0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.03.014
client proteins are involved in cell proliferation and malignant transformation, HSP90 inhibitors have clinical potential in cancer management. Two GA derivatives, namely, 17-allylamino-17-demethoxygeldanamycin (17-AAG) and 17-demethoxy-17-N,N-dimethylaminoethylamino-geldanamycin (17-DMAG), are currently implemented in clinical trials for the treatment of cancer [17–19]. This communication describes the production and characterization of a GA-specific antibody, which also recognizes other BAs such as HA, 17-AAG, and 17-DMAG.
Materials and methods Materials. Radicicol, herbimycin A, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), and glutaraldehyde were obtained from Sigma (St. Louis, MO), while Affi-Gel 10 resin was obtained from Bio-Rad (Hercules, CA), GA was kindly provided by
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E. Barzilay et al. / Biochemical and Biophysical Research Communications 330 (2005) 561–564
D. Newman of the National Cancer Institute (Rockville, MD). 17AAG and 17-DMAG were obtained from Kosan Biosciences (Hayward, CA). 17-Hexamethylenediamine derivatization of GA. 17-Hexamethylenediamine derivatization was performed essentially as described [7] to generate a GA derivative that can be covalently immobilized via its amino moiety. GA (10 mM in CHCl3) was reacted with 1,6-hexanediamine at 1:10 molar ratio, respectively, for 2 h in the dark, to generate quantitatively a single derivative, the required 17-hexamethylenediamine-17-demethoxygeldanamycin (HDG-GA, Fig. 1). HDG-GA was dissolved in DMSO to obtain a stock solution of 10 mg/ml. The chemical structure of the product was verified by NMR analysis. NMR (500 MHz, CDCl3) 9.20brs(NH), 7.28 s, 6.95d (J = 11 Hz), 6.58t (11 Hz), 6.25 m, 5.86 m (2H), 5.19 s, 4.80 m (NH2) 4.31d (J = 10), 3.57 m, 3.45 m, 3.63 s (OMe), 3.27 s (OMe), 3.02 m, 2.66 m (2H), 2.30 m, 2.04 s (CH3), 1.79 s (CH3), and 1.70 m. Conjugation of HDG-GA. Carrier proteins (KLH or BSA) were dissolved in double-distilled water (DDW) to a concentration of 10 mg/ml. The protein solution was mixed with HDG-GA at a volume ratio of 2:1, respectively. The mixture was titered to a pH of 8.5 with NaOH. Glutaraldehyde (1% in DDW) was added to the GA-carrier protein mixture at a volume ratio of 2:3, respectively. The mixture was incubated at room temperature in the dark for 24 h, followed by dialysis against phosphate-buffered saline (PBS), pH 7.4. Immobilization of HDG-GA on Affi-Gel 10 resin (Bio-Rad) was performed according to the manufacturerÕs instructions. Affi-Gel beads were washed with 5 bed volumes of cold isopropanol. Beads were then incubated with HDG-GA (1 bed volume, 5 mg/ml in DMSO) for 4 h in the dark at room temperature, followed by blocking in 100 mM ethanolamine (pH 7.5). Antibody production. Anti-GA antiserum was obtained by immunization of rabbits with the KLH-GA conjugate. For the first injection, 0.5 mg of KLH-GA conjugate (calculated on the basis of KLH content) in 0.5 ml PBS was emulsified (1:1) with complete FreundÕs adjuvant (Difco, Detroit, MI) and injected subcutaneously. Four booster injections of 0.5 mg KLH-GA conjugate emulsified with incomplete FreundÕs adjuvant were performed at three-week intervals.
The serum was subjected to affinity purification using the immobilized HDG-GA beads. Western blots and immunoblot inhibition assays. Western blotting was performed essentially as described [20]. For immunoblot inhibition assays, BSA-GA conjugate (1 lg/lane) was separated on 7.5% SDS– PAGE and transferred to a nitrocellulose membrane. The anti-GA antiserum (1:1000 in 5% skim milk) was incubated for 1 h at room temperature with GA (5 lM), prior to incubation with the nitrocellulose membrane. ELISA. Ninety-six-well plates were coated with 0.2 lg/well BSAGA. The affinity purified anti-GA antibody (50 ng/ml in 5% skim milk) was incubated with the inhibitory drug for 1 h at room temperature and was subsequently used for probing the BSA-GA-coated wells. Wells were then incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody. Detection was performed at 450 nm.
Results and discussion Generation of an anti-GA antibody To generate an antibody that would specifically recognize GA and its 17 position derivatives (Fig. 1, R1), we conjugated HDG-GA via its 17 position to the carrier protein. We rationalized that this mode of conjugation would render the remaining sites of GA accessible to antibody binding and thus allow the antibody to recognize GA and its 17-position derivatives, 17-AAG and 17-DMAG. The anti-GA antiserum was obtained by immunization of rabbits with the KLH-GA conjugate and was subjected to affinity purification using the immobilized HDG-GA beads. The anti-GA antibody recognizes both conjugated and free GA
Fig. 1. Schematic structure of benzoquinone ansamycins employed in this study. Geldanamycin (GA), herbimycin A (HA), 17-allylamino17-demethoxygeldanamycin (17-AAG), 17-demethoxy-17-N,N-dimethylaminoethylamino-geldanamycin (17-DMAG), and 17-hexamethylenediamine-17-demethoxygeldanamycin (HDG-GA).
To assess whether the antibody recognizes GA, we employed the BSA-conjugated GA. The BSA-GA conjugate was separated on 7.5% SDS–PAGE and transferred to a nitrocellulose membrane. BSA that was subjected to the same conditions as for conjugation was used as a control. Subsequently, the membrane was probed with the crude anti-GA antiserum (1:1000 in 5% skim milk). The results depicted in Fig. 2 (left panel) demonstrate that the antiserum specifically recognizes the BSA-GA conjugate. Next, we questioned whether the antiserum could recognize free, unconjugated GA. To address this, the anti-GA antiserum was incubated for 1 h at room temperature in the presence of GA (5 lM), prior to incubation with BSA-GA or mock-conjugated BSA blotted onto a nitrocellulose membrane. Pre-incubation of the anti-GA antiserum with free GA reduced antibody binding to background levels (Fig. 2, middle panel), thus demonstrating that the anti-GA antiserum recognizes free GA. GA-recognition by the affinity purified anti-GA antibody was also demonstrated by its reactivity with BSA-GA in Western blot analysis (Fig. 2, right panel).
E. Barzilay et al. / Biochemical and Biophysical Research Communications 330 (2005) 561–564
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Fig. 3. Recognition of different BAs by the anti-GA antibody. The anti-GA antibody (50 ng/ml in 5% skim milk) was incubated with the inhibitory drug for 1 h at room temperature and was subsequently used for probing ELISA plates coated with the BSA-GA conjugate (0.2 lg/well). The data are represented as percent of control-uninhibited binding of the anti-GA antibody to BSA-GA. Fig. 2. Binding of the anti-GA antibody to BSA-GA. GA conjugated to BSA and mock-conjugated BSA (1 lg/lane) were separated on 7.5% SDS–PAGE and transferred onto nitrocellulose membrane filters. The membrane filters were subsequently probed with the anti-GA antiserum (1:1000, left panel), anti-GA antiserum that was pre-incubated with GA (5 lM, middle panel) or the affinity purified anti-GA antibody (50 ng/ml, right panel). The BSA-GA conjugate (solid arrowhead) is accompanied by BSA-GA dimers (empty arrowhead) and high-order complexes, due to the glutaraldehyde-conjugation process. A similar pattern is observed in the mock-conjugated BSA.
[17–19] emphasizes the importance of the anti-GA antibody we have produced as it, or refined versions of it, may now be considered as powerful tools for monitoring the levels of the drug in the plasma of BA treated patients and for assessment of pharmacokinetics and pharmacodynamics of these drugs. Availability of the antibodies would thus be of particular importance for furthering the ongoing clinical studies.
Acknowledgments The anti-GA antibody recognizes other benzoquinone ansamycins We then raised the question of whether the anti-GA antibody recognizes BAs other than GA. Towards this end, we used the ELISA technique to determine the capacity of various BAs to inhibit binding of the antiGA antibody to BSA-GA-coated plates. The inhibitory compounds (GA, HA, 17-AAG or 17-DMAG) at concentrations ranging from 0.03 to 30 lM were added to the affinity purified anti-GA antibody (50 ng/ml in 5% skim milk) prior to its addition to the BSA-GA-coated wells. As a control we employed radicicol, a non-BA HSP90 inhibitor. Fig. 3 demonstrates that while radicicol did not interfere with antibody binding to the plates, all the BAs used in this assay inhibited antibody binding in a dose dependent manner. Moreover, all BAs examined were equally potent in inhibiting antibody binding to the BSA-GA-coated plates. The fact that HA was as potent as GA and as its 17-position derivatives in inhibiting binding of antibody to immobilized GA (Fig. 3) suggests that not all positions in the GA molecule take part in the antigenic determinants recognized by the anti-GA antibody. The antibody thus has a significant potential for further research on these drugs. Moreover, introduction of 17-AAG and 17-DMAG to clinical trials
This work was supported by a grant from the Israel Science Foundation administered by the Israel Academy of Sciences and Humanities (574/99 18.2) to D.N. This work was carried out in partial fulfillment of the requirements for the Ph.D. degree of Eran Barzilay from the Sackler Faculty of Medicine, Tel-Aviv University, Israel.
References [1] W.B. Pratt, The hsp90-based chaperone system: involvement in signal transduction from a variety of hormone and growth factor receptors, Proc. Soc. Exp. Biol. Med. 217 (1998) 420–434. [2] L. Neckers, T.W. Schulte, E. Mimnaugh, Geldanamycin as a potential anti-cancer agent: its molecular target and biochemical activity, Invest. New Drugs 17 (1999) 361–373. [3] L. Sepp-Lorenzino, Z. Ma, D.E. Lebwohl, A. Vinitsky, N. Rosen, Herbimycin A induces the 20S proteasome- and ubiquitindependent degradation of receptor tyrosine kinases, J. Biol. Chem. 270 (1995) 16580–16587. [4] M. Sakagami, P. Morrison, W.J. Welch, Benzoquinoid ansamycins (herbimycin A and geldanamycin) interfere with the maturation of growth factor receptor tyrosine kinases, Cell Stress Chaperones 4 (1999) 19–28. [5] P. Miller, C. DiOrio, M. Moyer, R.C. Schnur, A. Bruskin, W. Cullen, J.D. Moyer, Depletion of the erbB-2 gene product p185 by benzoquinoid ansamycins, Cancer Res. 54 (1994) 2724–2730.
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