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Fatty Acid Binding Proteins Expressed at the Human Blood–Brain Barrier Bind Drugs in an Isoform-Specific Manner

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ABSTRACT

Purpose

To examine the expression of fatty acid binding proteins (FABPs) at the human blood–brain barrier (BBB) and to assess their ability to bind lipophilic drugs.

Methods

mRNA and protein expression of FABP subtypes in immortalized human brain endothelial (hCMEC/D3) cells were examined by RT-qPCR and Western blot, respectively. FABPs that were found in hCMEC/D3 cells (hFABPs) were recombinantly expressed and purified from Escherichia coli C41(DE3) cells. Drug binding to these hFABPs was assessed using a fluorescence assay, which measured the ability of a panel of lipophilic drugs to displace the fluorescent probe compound 1-anilinonaphthalene-8-sulfonic acid (ANS).

Results

hFABP3, 4 and 5 were expressed in hCMEC/D3 cells at the mRNA and protein level. The competitive ANS displacement assay demonstrated that, in general, glitazones preferentially bound to hFABP5 (Ki: 1.0–28 μM) and fibrates and fenamates preferentially bound to hFABP4 (Ki: 0.100–17 μM). In general, lipophilic drugs appeared to show weaker affinities for hFABP3 relative to hFABP4 and hFABP5. No clear correlation was observed between the molecular structure or physicochemical properties of the drugs and their ability to displace ANS from hFABP3, 4 and 5.

Conclusions

hFABP3, 4 and 5 are expressed at the human BBB and bind differentially to a diverse range of lipophilic drugs. The unique expression and binding patterns of hFABPs at the BBB may therefore influence drug disposition into the brain.

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Abbreviations

ANS:

1-anilinonaphthalene-8-sulfonic acid

BBB:

Blood–brain barrier

BCECs:

Brain capillary endothelial cells

FABP:

Fatty acid binding protein

hCMEC/D3:

Human immortalized brain endothelial cells

REFERENCES

  1. Abbott NJ, Patabendige AA, Dolman DE, Yusof SR, Begley DJ. Structure and function of the blood-brain barrier. Neurobiol Dis. 2010;37:13–25.

    Article  CAS  PubMed  Google Scholar 

  2. Banks W. Drug transport into the central nervous system: using newer findings about the blood–brain barriers. Drug Deliv Transl Res. 2012;2:152–9.

    Article  CAS  PubMed  Google Scholar 

  3. Loscher W, Potschka H. Role of drug efflux transporters in the brain for drug disposition and treatment of brain diseases. Prog Neurobiol. 2005;76:22–76.

    Article  PubMed  Google Scholar 

  4. Pajouhesh H, Lenz GR. Medicinal chemical properties of successful central nervous system drugs. NeuroRx. 2005;2:541–53.

    Article  PubMed Central  PubMed  Google Scholar 

  5. Urquhart BL, Kim RB. Blood-brain barrier transporters and response to CNS-active drugs. Eur J Clin Pharmacol. 2009;65:1063–70.

    Article  CAS  PubMed  Google Scholar 

  6. Martinez MN, Amidon GL. A mechanistic approach to understanding the factors affecting drug absorption: a review of fundamentals. J Pharmacol Clin Toxicol. 2002;42:620–43.

    CAS  Google Scholar 

  7. Pardridge WM. Drug transport across the blood-brain barrier. J Cereb Blood Flow Metab. 2012;32:1959–72.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  8. Pardridge WM. The blood-brain barrier: bottleneck in brain drug development. NeuroRx. 2005;2:3–14.

    Article  PubMed Central  PubMed  Google Scholar 

  9. Velkov T, Horne J, Laguerre A, Jones E, Scanlon MJ, Porter CJ. Examination of the role of intestinal fatty acid-binding protein in drug absorption using a parallel artificial membrane permeability assay. Chem Biol. 2007;14:453–65.

    Article  CAS  PubMed  Google Scholar 

  10. Velkov T. Thermodynamics of lipophilic drug binding to intestinal fatty acid binding protein and permeation across membranes. Mol Pharm. 2009;6:557–70.

    Article  CAS  PubMed  Google Scholar 

  11. Luxon BA, Milliano MT. Cytoplasmic transport of fatty acids in rat enterocytes: role of binding to fatty acid-binding protein. Am J Physiol. 1999;277(2 Pt 1):G361–6.

    CAS  PubMed  Google Scholar 

  12. Zimmerman AW, Veerkamp JH. New insights into the structure and function of fatty acid-binding proteins. Cell Mol Life Sci. 2002;59:1096–116.

    Article  CAS  PubMed  Google Scholar 

  13. Bass NM, Manning JA. Tissue expression of three structurally different fatty acid binding proteins from rat heart muscle, liver, and intestine. Biochem Biophys Res Commun. 1986;137:929–35.

    Article  CAS  PubMed  Google Scholar 

  14. Hohoff C, Borchers T, Rustow B, Spener F, van Tilbeurgh H. Expression, purification, and crystal structure determination of recombinant human epidermal-type fatty acid binding protein. Biochemistry. 1999;38:12229–39.

    Article  CAS  PubMed  Google Scholar 

  15. He Y, Yang X, Wang H, Estephan R, Francis F, Kodukula S, et al. Solution-state molecular structure of apo and oleate-liganded liver fatty acid-binding protein. Biochemistry. 2007;46:12543–56.

    Article  CAS  PubMed  Google Scholar 

  16. Thompson J, Reese-Wagoner A, Banaszak L. Liver fatty acid binding protein: species variation and the accommodation of different ligands. Biochim Biophys Acta. 1999;23:1117–30.

    Google Scholar 

  17. Chuang S, Velkov T, Horne J, Porter CJ, Scanlon MJ. Characterization of the drug binding specificity of rat liver fatty acid binding protein. J Med Chem. 2008;51:3755–64.

    Article  CAS  PubMed  Google Scholar 

  18. Trevaskis NL, Nguyen G, Scanlon MJ, Porter CJ. Fatty acid binding proteins: potential chaperones of cytosolic drug transport in the enterocyte? Pharm Res. 2011;28:2176–90.

    Article  CAS  PubMed  Google Scholar 

  19. Velkov T, Lim ML, Horne J, Simpson JS, Porter CJ, Scanlon MJ. Characterization of lipophilic drug binding to rat intestinal fatty acid binding protein. Mol Cell Biochem. 2009;326:87–95.

    Article  CAS  PubMed  Google Scholar 

  20. Liu JW, Almaguel FG, Bu L, De Leon DD, De Leon M. Expression of E-FABP in PC12 cells increases neurite extension during differentiation: involvement of n-3 and n-6 fatty acids. J Neurochem. 2008;106:2015–29.

    PubMed Central  CAS  PubMed  Google Scholar 

  21. Mitchell RW, On NH, Del Bigio MR, Miller DW, Hatch GM. Fatty acid transport protein expression in human brain and potential role in fatty acid transport across human brain microvessel endothelial cells. J Neurochem. 2011;117:735–46.

    Article  CAS  PubMed  Google Scholar 

  22. Weksler BB, Subileau EA, Perriere N, Charneau P, Holloway K, Leveque M, et al. Blood-brain barrier-specific properties of a human adult brain endothelial cell line. FASEB J. 2005;19:1872–4.

    CAS  PubMed  Google Scholar 

  23. Froger A, Hall JE. Transformation of plasmid DNA into E. coli using the heat shock method. J Vis Exp. 2007;6:253.

    PubMed  Google Scholar 

  24. Cheng Y, Prusoff WH. Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem Pharmacol. 1973;22:3099–108.

    Article  CAS  PubMed  Google Scholar 

  25. Tjernberg A, Markova N, Griffiths WJ, Hallen D. DMSO-related effects in protein characterization. J Biomol Screen. 2006;11:131–7.

    Article  CAS  PubMed  Google Scholar 

  26. Sheng N, Li J, Liu H, Zhang A, Dai J. Interaction of perfluoroalkyl acids with human liver fatty acid-binding protein. Arch Toxicol. 2014;290:13895--906.

  27. Richieri GV, Ogata RT, Zimmerman AW, Veerkamp JH, Kleinfeld AM. Fatty acid binding proteins from different tissues show distinct patterns of fatty acid interactions. Biochemistry. 2000;39:7197–204.

    Article  CAS  PubMed  Google Scholar 

  28. Kleinfeld AM, Chu P, Romero C. Transport of long-chain native fatty acids across ipid bilayer membranes indicates that transbilayer flip-flop is rate limiting. Biochemistry. 1997;36:14146–58.

    Article  CAS  PubMed  Google Scholar 

  29. Patil R, Laguerre A, Wielens J, Headey SJ, Williams ML, Hughes ML, et al. Characterization of two distinct modes of drug binding to human intestinal fatty acid binding protein. ACS Chem Biol. 2014;9:2526–34.

    Article  CAS  PubMed  Google Scholar 

  30. Pelerin H, Jouin M, Lallemand MS, Alessandri JM, Cunnane SC, Langelier B, et al. Gene expression of fatty acid transport and binding proteins in the blood-brain barrier and the cerebral cortex of the rat: differences across development and with different DHA brain status. Prostaglandins Leukot Essent Fat Acids. 2014;91:213–20.

    Article  CAS  Google Scholar 

  31. Hagberg C, Mehlem A, Falkevall A, Muhl L, Eriksson U. Endothelial fatty acid transport: role of vascular endothelial growth factor B. Physiology. 2013;28:125–34.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  32. Bachmeier C, Mullan M, Paris D. Characterization and use of human brain microvascular endothelial cells to examine beta-amyloid exchange in the blood-brain barrier. Cytotechnology. 2010;62:519–29.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  33. Balendiran GK, Schnutgen F, Scapin G, Borchers T, Xhong N, Lim K, et al. Crystal structure and thermodynamic analysis of human brain fatty acid-binding protein. J Biol Chem. 2000;275:27045–54.

    CAS  PubMed  Google Scholar 

  34. Thompson J, Winter N, Terwey D, Bratt J, Banaszak L. The crystal structure of the liver fatty acid-binding protein. A complex with two bound oleates. J Biol Chem. 1997;272:7140–50.

    Article  CAS  PubMed  Google Scholar 

  35. Scapin G, Young AM, Kromminga A, Veerkamp J, Gordon J, Sacchettini J. High resolution X-ray studies of mammalian intestinal and muscle fatty acid-binding proteins provide an opportunity for defining the chemical nature of fatty acid: protein interactions. Mol Cell Biochem. 1993;123:3–13.

    Article  CAS  PubMed  Google Scholar 

  36. Hirose M, Sugiyama S, Ishida H, Niiyama M, Matsuoka D, Hara T, et al. Structure of the human-heart fatty-acid-binding protein 3 in complex with the fluorescent probe 1-ranilinonaphthalene-8-sulphonic acid. J Synchrotron Radiat. 2013;20:923–8.

  37. Gillilan RE, Ayers SD, Noy N. Structural basis for activation of fatty acid-binding protein 4. J Mol Biol. 2007;372:1246–60.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  38. Gutierrez-Gonzalez LH, Ludwig C, Hohoff C, Rademacher M, Hanhoff T, Ruterjans H, et al. Solution structure and backbone dynamics of human epidermal-type fatty acid-binding protein (E-FABP). Biochem J. 2002;364:725–37.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  39. Zimmerman AW, Rademacher M, Ruterjans H, Lucke C, Veerkamp JH. Functional and conformational characterization of new mutants of heart fatty acid-binding protein. Biochem J. 1999;344(Pt 2):495–501.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  40. Velkov T. Interactions between human liver fatty acid binding protein and peroxisome proliferator activated receptor selective drugs. PPAR Res. 2013; 938401.

  41. Kane CD, Bernlohr DA. A simple assay for intracellular lipid-binding proteins using displacement of 1-anilinonaphthalene 8-sulfonic acid. Anal Biochem. 1996;233:197–204.

    Article  CAS  PubMed  Google Scholar 

  42. Richieri GV, Ogata RT, Kleinfeld AM. Fatty acid interactions with native and mutant fatty acid binding proteins. Mol Cell Biochem. 1999;192:77–85.

    Article  CAS  PubMed  Google Scholar 

  43. Bergstrom CA, Charman SA, Nicolazzo JA. Computational prediction of CNS drug exposure based on a novel in vivo dataset. Pharm Res. 2012;29:3131–42.

    Article  PubMed  Google Scholar 

  44. Hughes ML, Liu B, Halls ML, Wagstaff KM, Patil R, Velkov T, et al. Fatty acid binding proteins 1 and 2 differentially modulate the activation of peroxisome proliferator-activated receptor alpha in a ligand-selective manner. J Biol Chem. 2015.[In Press]

  45. Tan NS, Shaw NS, Vinckenbosch N, Liu P, Yasmin R, Desvergne B, et al. Selective cooperation between fatty acid binding proteins and peroxisome proliferator-activated receptors in regulating transcription. Mol Cell Biol. 2002;22(14):5114–27.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

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ACKNOWLEDGMENTS AND DISCLOSURES

The ANZ Trustees (William Buckland Foundation) and the Australian Research Council (DP120102930) are acknowledged for their financial support of this project.

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Authors and Affiliations

  1. Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences,, Monash University, 381 Royal Parade, Parkville, Victoria, 3052, Australia

    Gordon S. Lee, Katharina Kappler, Christopher J. H. Porter & Joseph A. Nicolazzo

  2. Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia

    Martin J. Scanlon

Authors
  1. Gordon S. Lee
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  2. Katharina Kappler
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  3. Christopher J. H. Porter
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  4. Martin J. Scanlon
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  5. Joseph A. Nicolazzo
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Correspondence to Joseph A. Nicolazzo.

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Lee, G.S., Kappler, K., Porter, C.J.H. et al. Fatty Acid Binding Proteins Expressed at the Human Blood–Brain Barrier Bind Drugs in an Isoform-Specific Manner. Pharm Res 32, 3432–3446 (2015). https://doi.org/10.1007/s11095-015-1764-5

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  • DOI: https://doi.org/10.1007/s11095-015-1764-5

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