Human pregnane X receptor (PXR) is of vital importance in pharmaceutical and exogenous compound metabolism within the body. This provides strong motivation for investigating this orphan receptor’s activation by various pharmaceuticals, xenobiotics, and endocrine disrupting chemicals (EDCs). A nanomechanical transducer is developed to study xenobiotic and EDC interactions with the bioreceptor PXR’s ligand binding domain (LBD). The combination of immobilized LBD PXR with a nanostructured microcantilever (MC) platform allows for the sensitive, label-free study of ligand interaction with the receptor. PXR shows real-time, reversible responses when exposed to a specific pharmaceutical, EDCs, and xenobiotic ligands. Three EDCs binding interactions are tested, which include phthalic acid, nonylphenol, and bisphenol A, with PXR. PXR LBD was exposed to rifampicin, a potent PXR activator, with various injection and recovery times to study their interaction. A two protein array of PXR and estrogen receptor ? (ER-?) directly compares the nanomechanical responses of these receptors with rifampicin on a single platform.
KeywordsMicrocantileversNanobiosensingPregnane X receptorEstrogen receptorEndocrine disrupting chemicalsBioarray
[1] D. R. Nelson, L. Koymans, T. Kamataki, J. J. Stegeman, R. Feyereisen, D. J. Waxman, M. R. Waterman, O. Gotoh, M. J. Coon, R. W. Estabrook, I. C. Gunsalus and D. W. Nebert, “P450 Superfamily: Update on New Sequences, Gene Mapping, Ac-cession Numbers and Nomenclature,” Pharmacogenetics, Vol. 6, 1996, pp. 1-42. doi:10.1097/00008571-199602000-00002
[2] S. A. Kliewer, J. M. Lehmann and T. M. Willson, “Orphan Nu-clear Receptors: Shifting Endocrinology into Reverse,” Science, Vol. 284, 1999, pp. 757-760. doi:10.1126/science.284.5415.757
[3] E. L. Michalets, “Update: Clinically Significant Cyto-Chrome P-450 Drug Interactions,” Pharmacotherapy, Vol. 18, 1998, pp. 84-112.
[4] F. P. Guengerich, in: P. R. Ortiz de Montellano (Eds.), “Cyto-Chrome P450: Structure, Mechanism and Biochemistry,” Ple-num Publishers, 2005, pp. 377-425.
[5] J. Zhang, P. Kuehl, E. D. Green, J. W. Touchman, P. B. Wat-kins, A. Daly, S. D. Hall, P. Maurel, M. Relling, C. Brimer, K. Yasuda, S. A. Wrighton, M. Hancock, R. B. Kim, S. Strom, K. Thummel, C. G. Russell, J. R. Hudson, Jr., E. G. Schuetz and M. S. Boguski, “The Human Pregnane X Receptor: Genomic Structure and Identification and Functional Characterization of Natural Allelic Variants,” Pharmacogenetics, Vol. 11, 2001, pp. 555-572. doi:10.1097/00008571-200110000-00003
[6] S. A. Kliewer, J. T. Moore, L. Wade, J. L. Staudinger, M. A. Watson, S. A. Jones, D. D. McKee, B. B. Oliver, T. M. Will-son, R. H. Zetterstr?m, T. Perlmann and J. M. Lehmann, “An Orphan Nuclear Receptor Activated by Pregnanes Defines a Novel Steroid Signaling Pathway,” Cell, 92, 1998, pp. 73-82. doi:10.1016/S0092-8674(00)80900-9
[7] B. Blumberg, W. Sabbagh, Jr., H. Juguilon, J. Bolado, Jr., C. M. van Meter, E. S. Ong and R. M. Evans, “SXR, a Novel Steroid and Xenobiotic-Sensing Nuclear Receptor,” Genes and Development, Vol. 12, 1998, pp. 3195-3205. doi:10.1101/gad.12.20.3195
[8] G. Bertilsson, J. Heidrich, K. Svensson, M. ?sman, L. Jende-berg, M. Sydow-B?ckman, R. Ohlsson, H. Postlind, P. Blom-quist and A. Berkenstam, “Identification of a Human Nuclear Receptor Defines a New Signaling Pathway for Cyp3a Induc-tion,” Proceedings of the National Academy of Sciences, Vol. 95, 1998, pp. 12208-12213. doi:10.1073/pnas.95.21.12208
[9] R. E. Watkins, G. B. Wisely, L. B. Moore, J. L. Collins, M. H. Lambert, S. P. Williams, T. M. Willson, S. A. Kliewer and M. R. Redinbo, “The Human Nuclear Xenobiotic Receptor PXR: Structural Determinants of Directed Promiscuity,” Science, Vol. 292, 2001, pp. 2329-2333. doi:10.1126/science.1060762
[10] L. B. Moore, J. M. Maglich, D. D. McKee, B. Wisely, T. M. Willson, S. A. Kliewer, M. H. Lambert and J. T. Moore, “Pregnane X Receptor (PXR), Constitutive Androstane Re-ceptor (CAR) and Benzoate X Receptor (BXR) Define Three Pharmacologically Distinct Classes of Nuclear Receptors,” Molecular Endocrinology, Vol. 16, 2002, pp. 977-986. doi:10.1210/me.16.5.977
[11] C. Zhou, S. Verma and B. Blumberg, “The Steroid and Xeno-biotic Receptor (Sxr), Beyond Xenobiotic Metabolism,” Nu-clear Receptor Signaling, Vol. 7, 2009, pp. 1-21.
[12] S. A. Kliewer, B. Goodwin and T. M. Willson, “The Nuclear Pregnane X Receptor: A Key Regulator of Xenobiotic Me-tabolism,” Endocrine Reviews, Vol. 23, 2002, pp. 687-702. doi:10.1210/er.2001-0038
[13] J. E. Chrencik, J. Orans, L. B. Moore, Y. Xue, L. Peng, J. L. Collins, G. B. Wisely, M. H. Lambert, S. A. Kliewer and M. R. Redinbo, “Structural Disorder in the Complex of Human Pregnane X Receptor and the Macrolide Antibiotic Rifam-picin,” Molecular Endocrinology, Vol. 19, 2005, pp. 1125- 1134. doi:10.1210/me.2004-0346
[14] J. T. Moore and S. A. Kliewer, ‘Use of the Nuclear Recaptor Pxr to Predict Drug Interactions,” Toxicology, Vol. 153, 2000, pp. 1-10. doi:10.1016/S0300-483X(00)00300-0
[15] P. Dutta, K. Hill, P. G. Datskos and M. J. Sepaniak, “Devel-opment of a Nanomechanical Biosensor for Analysis of Endo-crine Disrupting Chemicals,” Lab Chip, Vol. 7, 2007, pp. 1184-1191. doi:10.1039/b704723a
[16] K. Hill, P. Dutta, A. Zareba, M. L. Eldridge and M. J. Sepaniak, “Morphological and Chemical Optimization of Microcantile-ver Surfaces for Thyroid System Sensing and Beyond,” Ana-lytica Chimica Acta, Vol. 625, 2008, pp. 55-62. doi:10.1016/j.aca.2008.07.005
[17] C. A. Tipple, N. V. Lavrik, M. Culha, J. Headrick, P. Datskos and M. J. Sepaniak, “Nanostructured Microcantilevers with Functionalized Cyclodextrin Receptor Phases: Self Assembled Monolayers and Vapor Deposited Films,” Analytical Chemis-try, Vol. 74, 2002, pp. 3118-3126. doi:10.1021/ac020074o
[18] P. Dutta, P. J. Chapman, P. G. Datskos and M. J. Sepaniak, “Characterization of Ligand-Functionalized Microcantilevers for Metal Ion Sensing,” Analytical Chemistry, Vol. 77, 2005, pp. 6601-6608. doi:10.1021/ac051082i
[19] P. Dutta, J. Sanseverino, P. G. Datskos and M. J. Sepaniak, “Detection of Cytokine Using a Microcantilever Biosensor,” NanoBiotechnology, 1, 2005, pp. 237-244. doi:10.1385/NBT:1:3:237
[20] R. Berger, E. Delamarche, H. P. Lang, C. Gerber, J. K. Gim-zewski, E. Meyer and H. J. Guntherodt, “Surface Stress in the Self-Assembly of Alkanethiols on Gold,” Science, Vol. 276, 1997, pp. 2021-2024. doi:10.1126/science.276.5321.2021
[21] T. Thundat, E. A. Wachter, S. L. Sharp and R. J. Warmack, “Detection of Mercury Vapor Using Resonating Microcanti-levers,” Applied Physics Letters, Vol. 66, 1995, pp. 1695-1697. doi:10.1063/1.113896
[22] P. Dutta, C. A. Tipple, N. V. Lavrik, P. G. Datskos, O. Hof-stetter and M. J. Sepaniak, “Enantioselective Sensors Based on Antibody Mediated Nanomechanics,” Analytical Chemistry, Vol. 75, 2003, pp. 2342-2348. doi:10.1021/ac034031z
[23] G. Wu, R. H. Datar, K. M. Hansen, T. Thundat, R. J. Cote and A. Majumdar, “Bioassay of Prostate-Specific Antigen (Psa) Using Microcantilevers,” National Biotechnology, Vol. 19, 2001, pp. 856-860. doi:10.1038/nbt0901-856
[24] C. Grogan, R. Raiteri, G. M. O’Connor, T. J. Glynn, V. Cun-ningham, M. Kane, M. Charlton and D. Leech, “Characteriza-tion of an Antibody Coated Microcantilever as a Potential Immuno-Based Biosensor,” Biosensor Bioelectronics, Vol. 17, 2002, pp. 201-207. doi:10.1016/S0956-5663(01)00276-7
[25] J. Fritz, M. K. Baller, H. P. Lang, H. Rothuizen, P. Vettiger, E. Meyer, H. Guntherodt, C. Gerber and J. K. Gimzewski, “Translating Biomolecular Recognition into Nanomechanics,” Science, Vol. 288, 2000, pp. 316-318. doi:10.1126/science.288.5464.316
[26] G. Shekhawat, S. H. Tark and V. P. Dravid, “MOS-FET-Embedded Microcantilevers for Measuring Deflection in Biomolecular Sensors,” Science, Vol. 311, 2006, pp. 1592- 1595. doi:10.1126/science.1122588
[27] S. A. Jones, L. B. Moore, J. L. Shenk, G. B. Wisely, G. A. Hamilton, .D. D. McKee, N. C. O. Tomkinson, E. L. LeCluyse, M. H. Lambert, T. M. Willson, S. A. Kliewer and J. T. Moore, “The Pregnane X Receptor: A Promis-Cuous Xenobiotic Re-ceptor That Has Diverged during Evolution,” Molecular En-docrinology, Vol. 14, 2000, pp. 27-39. doi:10.1210/me.14.1.27
[28] S. J. Shukla, D. Nguyen, R. MacArthur, A. Simeonov, W. J. Frazee, T. M. Hallis, B. D. Marks, U. Singh, H. C. Eliason, J. Printen, C. P. Austin, J. Inglese and D. S. Auld, “Identification of Pregnane X Receptor Ligands Using Time-Resolved Fluo-rescence Resonance Energy Transfer and Quantitative High- Throughput Screening,” Assay and Drug Development Tech-nologies, Vol. 7, 2009, pp. 143-169. doi:10.1089/adt.2009.193
[29] G. G. Stoney, “The Tension of Metallic Films Deposited by Electrolysis,” Proceeding of the Royal Society of London, Se-rial A, Vol.82, 1909, pp. 172-177.
[30] T. S. Kim, D. S. Yoon and J. H. Lee, in: N. P. Mahalik, Ed. “Micromanufacturing and Nanotechnology,” Springer, Berlin, 2006, pp. 299-321.
[31] N. V. Lavrik, C. A. Tipple, M. J. Sepaniak and P. G. Datskos, “Enhanced Chemi-Mechanical Transduction at Nanostructured Interfaces,” Chemical Physics Letters, Vol. 336, 2001, pp. 371-376. doi:10.1016/S0009-2614(01)00155-5
[32] J. R. Premkumar, O. Lev, R. S. Marks, B. Polyak, R. Rosen and S. Belkin, “Antibody-based Immobilization Of Biolumi-nescent Bacterial Sensor Cells,” Talanta, Vol. 55, 2001, pp. 1029-1038. doi:10.1016/S0039-9140(01)00533-1
[33] S. W. Park, Y. I. Kim, K. H. Chung, S. I. Hong and S. W. Kim, “Covalent Immobilization of Gl-7-Aca Acylase on Silica Gel Through Silanization,” Reactive Functional Polymers, Vol. 51, 2002, pp. 79-92. doi:10.1016/S1381-5148(02)00028-7
[34] P. J. Chapman, Z. Long, P. G. Datskos, R. Archibald and M. J. Sepaniak, “Ligand-Functionalized Microcantilever Arrays for Metal Ion Sensing,” Analytical Chemistry, Vol. 79, 2007, pp. 7062-7068. doi:10.1021/ac070754x
[35] L.R. Senesac, P. Dutta, P. G. Datskos and M.J. Sepaniak, “Analyte Identification Using Differentially-Functionalized Microcantilever Arrays and Artificial Neural Nets,” Analytical Chemistry Acta, Vol. 558, 2006, pp. 94-101. doi:10.1016/j.aca.2005.11.024
[36] P.J. Chapman, Z. Long, P.G. Datskos, R. Archbald and M.J. Sepaniak, “Ligand-Functionalized Microcantilever Arrays for Metal Ion Sensing” Analytical Chemistry, Vol. 79, 2007, pp. 7062-7068. doi:10.1021/ac070754x
[37] Z. Long, John Story, Samuel Lewis and M.J. Sepaniak “Land-fill Siloxane Gas Sensing Using Differentiating, Responsive Phase Coated Microcantilever Arrays” Analytical Chemistry, Vol. 81, 2009, pp. 2575-2580. doi:10.1021/ac802494v
[38] M. J. Sepaniak, P. G. Datskos, N. V. Lavrik and C. Tipple, “Microcantilever Transducers: A New Approach in Sensor Technology,” Analytical Chemistry, 2002, 568A-575A.
[39] J. L. Staudinger, B. Goodwin, S. A. Jones, D. Hawkins-Brown, K. I. MacKenzie, A. LaTour, Y. Liu, C. D. Klaassen, K. K. Brown, J. Reinhard, T. M. Willson, B. H. Koller and S. A. Kliewer, “The Nuclear Receptor PXR is a Lithocholic Acid Sensor That Protects against Liver Toxicity,” Processings of the National Academy Sciences, Vol. 98, 2001, pp. 3369-3374. doi:10.1073/pnas.051551698
[40] E. Y. H. Yeung, T. Sueyoshi, M. Negishi and T. K. H. Chang, “Identification of Ginkgo Biloba as a Novel Activator of Pregnane X Receptor,” Drug Metabolism and Disposition, Vol. 36, 2008, pp. 2270-2276. doi:10.1124/dmd.108.023499
[41] S. Kortagere, D. Chekmarev, W. J. Welsh and S. Ekins, “Hy-brid Scoring and Classification Approaches to Predict Human Pregnane X Receptor Activators,” Pharmaceutical Research, Vol. 26, 2009, pp. 1001-1011. doi:10.1007/s11095-008-9809-7
[42] P. Li, “Evaluation of Luciferin-Isopropyl Acetal as a CYP3A4 Substrate for Human Hepatocytes: Effects of Organics Sol-vents, Cytochrome P450 (P450) Inhibitors and P450 Induc-ers,” Drug Metabolism and Disposition, Vol. 37, 2009, pp. 1598-1603. doi:10.1124/dmd.109.027268
[43] M. Abdelrahim, E. Ariazi, K. Kim, S. Khan, R. Barhoumi, R. Burghardt, S. Liu, D. Hill, R. Finnell, B. Wlodarczyk, V. C. Jordan and S. Safe, “3-Methylcholanthrene and Other Aryl Hydrocarbon Receptor Agonists Directly Activate Estrogen Receptor,” Cancer Research, Vol. 66, 2006, pp. 2459-2467. doi:10.1158/0008-5472.CAN-05-3132
[44] J. P. Jackson, K. K. Kabirov, I. M. Kapetanovic and A. Lyu-bimov, “In Vitro Assessment of p450 Induction Potential of Novel Chemopreventive Agents sr13668, 9-cis-uab30 and Pentamethychromanol In Primary Cultures of Human Hepa-tocytes,” Chemical-Biology Interactive, Vol. 179, 2009, pp. 263-272. doi:10.1016/j.cbi.2008.12.005
[45] H. Kuramitz, J. Natsui, K. Sugawara, S. Itoh and S. Tanaka, “Electrochemical Evaluation of the Interaction between Endo-crine Disruptor Chemicals and Estrogen Receptor Using 17-Estradiol Labeled with Daunomycin,” Analytical Chemistry, Vol. 74, 2002, pp. 533-538. doi:10.1021/ac010426b
[46] G. G. J. M. Kuiper, J. G. Lemmen, B. Carlson, J. C. Corton, S. H. Safe, P. T. Van Der Saag, B. Van Der Burg and J. Gustafs-son, “Interaction of Estrogenic Chemicals and Phytoestrogens with Estrogen Receptor,” Endocrinology, Vol. 139, 1998, pp. 4252-4263. doi:10.1210/en.139.10.4252
[47] S. Stoica, B. S. Katzenellenbogen and M. B. Martin, “Activa-tion of Estrogen Receptor—by the Heavy Metal Cadmium,” Molecular Endocrinology, Vol. 14, 2000, pp. 545-553. doi:10.1210/me.14.4.545
[48] D. J. Mangelsdorf, C. Thummel, M. Besto, P. Herrlich, G. Schütz, K. Umesono, B. Blumberg, P. Kastner, M. Mark, P. Chambon and R. M. Evans, “The Nuclear Receptor Super-family: The Second Decade,” Cell, Vol. 83, 1995, pp. 835-839. doi:10.1016/0092-8674(95)90199-X
[49] H. Masuyama, Y. Hiramatsu, M. Kunitomi, T. Kudo and P. N. MacDonald, “Endocrine Disrupting Chemicals, Phthalic Acid and Nonylphenol, Activate Pregnane X Receptor-Mediated Transcription,” Molecular Endocrinology, Vol. 14, 2000, pp. 421-428. doi:10.1210/me.14.3.421
[50] H. Masuyama, N. Suwaki, Y. Tateishi, H. Nakatsukasa, T. Seqawa and Y. Hiramatsu, “The Pregnane X Receptor Regu-lates Gene Expression in a Ligand and Promoter-Selective Fashion,” Molecular Endocrinology, Vol. 19, 2005, pp. 1170- 1180. doi:10.1210/me.2004-0434
[51] D. Hopwood, C. R. Allen and M. McCabe, “The Reactions between Glutaraldehyde and Various Protein. An Investigation of Their Kinetics,” Histochemical Journal, Vol. 2, 1970, pp. 137-150. doi:10.1007/BF01003541
[52] D. R. Walt and V. I. Agayn, “The Chemistry of Enzyme and Protein Immobilization with Glutaraldehyde,” Trends Analyti-cal Chemistry, Vol. 13, 1994, pp. 425-430. doi:10.1016/0165-9936(94)85023-2