Skip to main content

Advertisement

SpringerLink
Log in

Evaluating Fmoc-amino acids as selective inhibitors of butyrylcholinesterase

  • Original Article
  • Published:
Amino Acids Aims and scope Submit manuscript

Abstract

Cholinesterases are involved in neuronal signal transduction, and perturbation of function has been implicated in diseases, such as Alzheimer’s and Huntington’s disease. For the two major classes of cholinesterases, such as acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), previous studies reported BChE activity is elevated in patients with Alzheimer’s disease, while AChE levels remain the same or decrease. Thus, the development of potent and specific inhibitors of BChE have received much attention as a potential therapeutic in the alleviation of neurodegenerative diseases. In this study, we evaluated amino acid analogs as selective inhibitors of BChE. Amino acid analogs bearing a 9-fluorenylmethyloxycarbonyl (Fmoc) group were tested, as the Fmoc group has structural resemblance to previously described inhibitors. We identified leucine, lysine, and tryptophan analogs bearing the Fmoc group as selective inhibitors of BChE. The Fmoc group contributed to inhibition, as analogs bearing a carboxybenzyl group showed ~tenfold higher values for the inhibition constant (K I value). Inclusion of a t-butoxycarbonyl on the side chain of Fmoc tryptophan led to an eightfold lower K I value compared to Fmoc tryptophan alone suggesting that modifications of the amino acid side chains may be designed to create inhibitors with higher affinity. Our results identify Fmoc-amino acids as a scaffold upon which to design BChE-specific inhibitors and provide the foundation for further experimental and computational studies to dissect the interactions that contribute to inhibitor binding.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Abreu-Villaca Y, Filgueiras CC, Manhaes AC (2011) Developmental aspects of the cholinergic system. Behav Brain Res 221(2):367–378. doi:10.1016/j.bbr.2009.12.049

    Article  CAS  PubMed  Google Scholar 

  • Anand P, Singh B (2013) A review on cholinesterase inhibitors for Alzheimer’s disease. Arch Pharmacal Res 36(4):375–399. doi:10.1007/s12272-013-0036-3

    Article  CAS  Google Scholar 

  • Andersson CD, Forsgren N, Akfur C, Allgardsson A, Berg L, Engdahl C, Qian W, Ekstrom F, Linusson A (2013) Divergent structure-activity relationships of structurally similar acetylcholinesterase inhibitors. J Med Chem 56(19):7615–7624. doi:10.1021/jm400990p

    Article  CAS  PubMed  Google Scholar 

  • Augustinsson K (1948) On the specificity of cholinesterase. Biol Bull 95(2):241

    CAS  PubMed  Google Scholar 

  • Bar-On P, Millard CB, Harel M, Dvir H, Enz A, Sussman JL, Silman I (2002) Kinetic and structural studies on the interaction of cholinesterases with the anti-Alzheimer drug rivastigmine. Biochemistry 41(11):3555–3564

    Article  CAS  PubMed  Google Scholar 

  • Behrendt R, White P, Offer J (2016) Advances in Fmoc solid-phase peptide synthesis. J Pept Sci 22(1):4–27. doi:10.1002/psc.2836

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Berg L, Andersson CD, Artursson E, Hornberg A, Tunemalm AK, Linusson A, Ekstrom F (2011) Targeting acetylcholinesterase: identification of chemical leads by high throughput screening, structure determination and molecular modeling. PLoS One 6(11):e26039. doi:10.1371/journal.pone.0026039

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Brus B, Kosak U, Turk S, Pislar A, Coquelle N, Kos J, Stojan J, Colletier JP, Gobec S (2014) Discovery, biological evaluation, and crystal structure of a novel nanomolar selective butyrylcholinesterase inhibitor. J Med Chem 57(19):8167–8179. doi:10.1021/jm501195e

    Article  CAS  PubMed  Google Scholar 

  • Carolan CG, Dillon GP, Khan D, Ryder SA, Gaynor JM, Reidy S, Marquez JF, Jones M, Holland V, Gilmer JF (2010) Isosorbide-2-benzyl carbamate-5-salicylate, a peripheral anionic site binding subnanomolar selective butyrylcholinesterase inhibitor. J Med Chem 53(3):1190–1199. doi:10.1021/jm9014845

    Article  CAS  PubMed  Google Scholar 

  • Dori A, Soreq H (2006) ARP, the cleavable C-terminal peptide of “readthrough” acetylcholinesterase, promotes neuronal development and plasticity. J Mol Neurosci 28(3):247–255. doi:10.1385/jmn:28:3:247

    Article  CAS  PubMed  Google Scholar 

  • Dvir H, Silman I, Harel M, Rosenberry TL, Sussman JL (2010) Acetylcholinesterase: from 3D structure to function. Chem-Biol Interact 187(1–3):10–22. doi:10.1016/j.cbi.2010.01.042

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ellman GL, Courtney KD, Andres V Jr, Feather-Stone RM (1961) A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 7:88–95

    Article  CAS  PubMed  Google Scholar 

  • Fields GB, Noble RL (1990) Solid phase peptide synthesis utilizing 9-fluorenylmethoxycarbonyl amino acids. Int J Pept Protein Res 35(3):161–214

    Article  CAS  PubMed  Google Scholar 

  • Grisaru D, Sternfeld M, Eldor A, Glick D, Soreq H (1999) Structural roles of acetylcholinesterase variants in biology and pathology. Eur J Biochem 264(3):672–686

    Article  CAS  PubMed  Google Scholar 

  • Halliday AC, Greenfield SA (2012) From protein to peptides: a spectrum of non-hydrolytic functions of acetylcholinesterase. Protein Pept Lett 19(2):165–172

    Article  CAS  PubMed  Google Scholar 

  • Hartsel JA, Wong DM, Mutunga JM, Ma M, Anderson TD, Wysinski A, Islam R, Wong EA, Paulson SL, Li J, Lam PC, Totrov MM, Bloomquist JR, Carlier PR (2012) Re-engineering aryl methylcarbamates to confer high selectivity for inhibition of Anopheles gambiae versus human acetylcholinesterase. Bioorg Med Chem Lett 22(14):4593–4598. doi:10.1016/j.bmcl.2012.05.103

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hogan DB (2014) Long-term efficacy and toxicity of cholinesterase inhibitors in the treatment of Alzheimer disease. Can J Psychiatry 59(12):618–623

    PubMed  PubMed Central  Google Scholar 

  • Huang L, Su T, Li X (2013) Natural products as sources of new lead compounds for the treatment of Alzheimer’s disease. Curr Top Med Chem 13(15):1864–1878

    Article  CAS  PubMed  Google Scholar 

  • Jiang H, Zhang XJ (2008) Acetylcholinesterase and apoptosis. A novel perspective for an old enzyme. FEBS J 275(4):612–617. doi:10.1111/j.1742-4658.2007.06236.x

    Article  CAS  PubMed  Google Scholar 

  • Kaboudin B, Emadi S, Hadizadeh A (2009) Synthesis of novel phosphorothioates and phosphorodithioates and their differential inhibition of cholinesterases. Bioorg Chem 37(4):101–105. doi:10.1016/j.bioorg.2009.05.002

    Article  CAS  PubMed  Google Scholar 

  • Kamal MA, Qu X, Yu QS, Tweedie D, Holloway HW, Li Y, Tan Y, Greig NH (2008) Tetrahydrofurobenzofuran cymserine, a potent butyrylcholinesterase inhibitor and experimental Alzheimer drug candidate, enzyme kinetic analysis. J Neural Transm (Vienna) 115(6):889–898. doi:10.1007/s00702-008-0022-y

    Article  CAS  Google Scholar 

  • Kleywegt GJ, Jones TA (1994) Detection, delineation, measurement and display of cavities in macromolecular structures. Acta Crystallogr D Biol Crystallogr 50(Pt 2):178–185. doi:10.1107/s0907444993011333

    Article  CAS  PubMed  Google Scholar 

  • Law KS, Acey RA, Smith CR, Benton DA, Soroushian S, Eckenrod B, Stedman R, Kantardjieff KA, Nakayama K (2007) Dialkyl phenyl phosphates as novel selective inhibitors of butyrylcholinesterase. Biochem Biophys Res Commun 355(2):371–378. doi:10.1016/j.bbrc.2007.01.186

    Article  CAS  PubMed  Google Scholar 

  • Macdonald IR, Martin E, Rosenberry TL, Darvesh S (2012) Probing the peripheral site of human butyrylcholinesterase. Biochemistry 51(36):7046–7053. doi:10.1021/bi300955k

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Mallender WD, Szegletes T, Rosenberry TL (2000) Acetylthiocholine binds to asp74 at the peripheral site of human acetylcholinesterase as the first step in the catalytic pathway. Biochemistry 39(26):7753–7763

    Article  CAS  PubMed  Google Scholar 

  • Meshorer E, Erb C, Gazit R, Pavlovsky L, Kaufer D, Friedman A, Glick D, Ben-Arie N, Soreq H (2002) Alternative splicing and neuritic mRNA translocation under long-term neuronal hypersensitivity. Science 295(5554):508–512. doi:10.1126/science.1066752

    Article  CAS  PubMed  Google Scholar 

  • Mushtaq G, Greig NH, Khan JA, Kamal MA (2014) Status of acetylcholinesterase and butyrylcholinesterase in Alzheimer’s disease and type 2 diabetes mellitus. CNS Neurol Disord Drug Targets 13(8):1432–1439

    Article  CAS  PubMed  Google Scholar 

  • Musial A, Bajda M, Malawska B (2007) Recent developments in cholinesterases inhibitors for Alzheimer’s disease treatment. Curr Med Chem 14(25):2654–2679

    Article  CAS  PubMed  Google Scholar 

  • Nicolet Y, Lockridge O, Masson P, Fontecilla-Camps JC, Nachon F (2003) Crystal structure of human butyrylcholinesterase and of its complexes with substrate and products. J Biol Chem 278(42):41141–41147. doi:10.1074/jbc.M210241200

    Article  CAS  PubMed  Google Scholar 

  • Nizri E, Brenner T (2013) Modulation of inflammatory pathways by the immune cholinergic system. Amino Acids 45(1):73–85. doi:10.1007/s00726-011-1192-8

    Article  CAS  PubMed  Google Scholar 

  • Pagano G, Rengo G, Pasqualetti G, Femminella GD, Monzani F, Ferrara N, Tagliati M (2015) Cholinesterase inhibitors for Parkinson’s disease: a systematic review and meta-analysis. J Neurol Neurosurg Psychiatry 86(7):767–773. doi:10.1136/jnnp-2014-308764

    Article  PubMed  Google Scholar 

  • Pinho BR, Ferreres F, Valentao P, Andrade PB (2013) Nature as a source of metabolites with cholinesterase-inhibitory activity: an approach to Alzheimer’s disease treatment. J Pharm Pharmacol 65(12):1681–1700. doi:10.1111/jphp.12081

    Article  CAS  PubMed  Google Scholar 

  • Radic Z, Pickering NA, Vellom DC, Camp S, Taylor P (1993) Three distinct domains in the cholinesterase molecule confer selectivity for acetyl- and butyrylcholinesterase inhibitors. Biochemistry 32(45):12074–12084

    Article  CAS  PubMed  Google Scholar 

  • Riddles PW, Blakeley RL, Zerner B (1983) Reassessment of Ellman’s reagent. Methods Enzymol 91:49–60

    Article  CAS  PubMed  Google Scholar 

  • Saxena A, Redman AM, Jiang X, Lockridge O, Doctor BP (1997) Differences in active site gorge dimensions of cholinesterases revealed by binding of inhibitors to human butyrylcholinesterase. Biochemistry 36(48):14642–14651. doi:10.1021/bi971425+

    Article  CAS  PubMed  Google Scholar 

  • Segel IH (1974) Enzyme kinetics behavior and analysis of rapid equilibrium and steady-state enzyme systems. Wiley, New York

    Google Scholar 

  • Soreq H, Gnatt A, Loewenstein Y, Neville LF (1992) Excavations into the active-site gorge of cholinesterases. Trends Biochem Sci 17(9):353–358

    Article  CAS  PubMed  Google Scholar 

  • Sussman JL, Harel M, Frolow F, Oefner C, Goldman A, Toker L, Silman I (1991) Atomic structure of acetylcholinesterase from Torpedo californica: a prototypic acetylcholine-binding protein. Science 253(5022):872–879

    Article  CAS  PubMed  Google Scholar 

  • Szegletes T, Mallender WD, Thomas PJ, Rosenberry TL (1999) Substrate binding to the peripheral site of acetylcholinesterase initiates enzymatic catalysis. Substrate inhibition arises as a secondary effect. Biochemistry 38(1):122–133. doi:10.1021/bi9813577

    Article  CAS  PubMed  Google Scholar 

  • Vellom DC, Radic Z, Li Y, Pickering NA, Camp S, Taylor P (1993) Amino acid residues controlling acetylcholinesterase and butyrylcholinesterase specificity. Biochemistry 32(1):12–17

    Article  CAS  PubMed  Google Scholar 

  • Verma A, Wong DM, Islam R, Tong F, Ghavami M, Mutunga JM, Slebodnick C, Li J, Viayna E, Lam PC, Totrov MM, Bloomquist JR, Carlier PR (2015) 3-Oxoisoxazole-2(3H)-carboxamides and isoxazol-3-yl carbamates: resistance-breaking acetylcholinesterase inhibitors targeting the malaria mosquito, Anopheles gambiae. Bioorg Med Chem 23(6):1321–1340. doi:10.1016/j.bmc.2015.01.026

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wong DM, Li J, Lam PC, Hartsel JA, Mutunga JM, Totrov M, Bloomquist JR, Carlier PR (2013) Aryl methylcarbamates: potency and selectivity towards wild-type and carbamate-insensitive (G119S) Anopheles gambiae acetylcholinesterase, and toxicity to G3 strain An. gambiae. Chem Biol Interact 203(1):314–318. doi:10.1016/j.cbi.2012.09.001

    Article  CAS  PubMed  Google Scholar 

  • Xie R, Zhao Q, Zhang T, Fang J, Mei X, Ning J, Tang Y (2013) Design, synthesis and biological evaluation of organophosphorous-homodimers as dual binding site acetylcholinesterase inhibitors. Bioorg Med Chem 21(1):278–282. doi:10.1016/j.bmc.2012.10.030

    Article  CAS  PubMed  Google Scholar 

  • Zhao YH, Abraham MH, Zissimos AM (2003) Fast calculation of van der Waals volume as a sum of atomic and bond contributions and its application to drug compounds. J Org Chem 68(19):7368–7373. doi:10.1021/jo034808o

    Article  CAS  PubMed  Google Scholar 

  • Zhao Q, Xie R, Zhang T, Fang J, Mei X, Ning J, Tang Y (2011) Homo- and hetero-dimers of inactive organophosphorous group binding at dual sites of AChE. Bioorg Med Chem Lett 21(21):6404–6408. doi:10.1016/j.bmcl.2011.08.098

    Article  CAS  PubMed  Google Scholar 

  • Zhao T, Ding K-M, Zhang Z, Cheng Z-M, Wang C-H, Wang ZT (2013) Acetylcholinesterase and butyrylcholinesterase inhibitory activities of B-carboline and quinoline alkaloids derivatives from the plants of genus Peganum. J Chem 2013:1–6. doi:10.1155/2013/717232

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We thank the reviewers for helpful comments on this manuscript. This work was supported by startup funds provided by California State University, Long Beach. Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Numbers UL1GM118979 and RL5GM118978. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. NMR instrumentation was provided for by the National Science Foundation (MRI CHE-1337559). Any opinions, findings, and conclusions expressed are those of the author(s) and do not necessarily reflect the views of NSF.

Author information

Authors and Affiliations

  1. Department of Chemistry and Biochemistry, California State University, Long Beach, 1250 Bellflower Boulevard, Long Beach, CA, 90840-9507, USA

    Jeannette Gonzalez, Jennifer Ramirez & Jason P. Schwans

Authors
  1. Jeannette Gonzalez
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jennifer Ramirez
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jason P. Schwans
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jason P. Schwans.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Research involving human participants and/or animals

This article does not contain any studies with human participants or animals performed by any of the authors.

Additional information

Handling Editor: T. Langer.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOC 107 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Gonzalez, J., Ramirez, J. & Schwans, J.P. Evaluating Fmoc-amino acids as selective inhibitors of butyrylcholinesterase. Amino Acids 48, 2755–2763 (2016). https://doi.org/10.1007/s00726-016-2310-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00726-016-2310-4

Keywords

Search

Navigation