A potent and highly selective inhibitor of human α-1,3-fucosyltransferase via click chemistry

Article abstract of DOI:10.1021/ja0302836

Potent inhibitors of fucosyltransferases, and glycosyltransferases in general, have been elusive due to the inherent barriers surrounding the family of glycosyltransfer reactions. The problems of weak substrate affinity and low catalytic proficiency of fu

Full text of DOI:10.1021/ja0302836

Published on Web 07/22/2003
A Potent and Highly Selective Inhibitor of Human r-1,3-Fucosyltransferase
via Click Chemistry
Lac V. Lee, Michael L. Mitchell, Shih-Jung Huang, Valery V. Fokin,* K. Barry Sharpless,* and
Chi-Huey Wong*
Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute,
10550 Torrey Pines Road, La Jolla, California 92037
Received May 7, 2003; E-mail: wong@scripps.edu; sharples@scripps.edu; fokin@scripps.edu
Fucosyltransferases (Fuc-T) catalyze the final glycosylation step
in the biosynthesis and expression of many important saccharides,
such as sialyl Lewis x (sLex) and sialyl Lewis a (sLea) of cell-
surface glycoproteins and glycolipids. These and other fucosylated
oligosaccharide structures are central to cell-cell interactions and
cell migration in connection with physiological and pathological
processes such as fertilization, embryogenesis, lymphocyte traf-
ficking, immune responses, and cancer metastasis.1 The terminal
step in the biosynthetic pathway of these fucose-containing sac-
charides is the transfer of the L-fucose from guanosine diphosphate
â-L-fucose (GDP-fucose) to the corresponding glycoconjugate
acceptor.1
Different strategies have been employed to identify inhibitors
of Fuc-T. Most rely on the design of acceptor, donor, and transition-
state analogues.2-4 Several factors inherent to the chemistry of
glycosyl-transfer reactions make rational design of Fuc-T inhibitors
difficult. The enzymatic reaction involves a complex four-partner
transition-state (sugar donor, acceptor, divalent metal, and nucle-
Figure 1. Fucosyltransferase-catalyzed reaction. The fucose moiety is
otide). Measurement of the catalytic proficiency for GDP-fucose
transferred from GDP-fucose to an acceptor molecule, LacNAc-R. It is
hydrolysis shows that the Fuc-T has a low affinity for the transition-
state of the GDP-fucose moiety.5 The affinity for the acceptor
substrate is also low,5-7 and there is no structural data for Fuc-Ts.
Hence, the rational development of potent inhibitors for Fuc-Ts
has been difficult, and to date the best inhibitors are in the
micromolar range.2-4
As reported here, a substantially better Fuc-T inhibitor has been
found. Success came quickly, using the click chemistry approach,8-10
to rapidly identify a new optimal binding component. In fact, the
latest advance in this area, the Cu(I)-catalyzed triazole synthesis,10,11
made it possible to create the desired library of GDP-triazole
candidates in water, without protecting groups (even for dianionic
phosphate linkage), so that the crude aqueous reaction solutions
were pure enough to test “as is.”
evident from the inhibition (Ki) and apparent dissociation (Km) constants
that the majority of the binding energies exist at the GDP moiety and to
some extent the hydrophobic aglycon group.5,12 The design of the GDP-
triazole library takes advantage of the binding energies for the GDP and
hydrophobic aglycon moiety.
Scheme 1. Triazole Synthesis in Microtiter Plate for Screening in
Situ
Since the majority of the binding energy of Fuc-T for its
substrates lies at the GDP moiety4,5 (Figure 1), and the hydrophobic
pocket adjacent to the binding site of the acceptor molecule
enhances affinity of the acceptor molecule by 70-fold,12 we have
designed a library of compounds which retained the important GDP
core, while the attached hydrophobic group and the linker length
were varied. The carbohydrate moieties on the donor and acceptor
substrates were omitted to simplify the synthetic challenge. An
inhibitor that is derived from the screening of this library would
constitute the optimal linker and hydrophobic moiety, and would
result in the specificity for the target enzyme.
Thus, 85 azide compounds were synthesized, representing the
diversity in the hydrophobic group and the tether (2 to 6 carbons;
see Supporting Information). Using the new copper-catalyzed
process, each of these azide molecules was linked to the GDP-
alkyne core to give the 85 triazole candidates (Scheme 1). The GDP-
triazole compounds were screened for inhibitory activity directly
in microtiter plates, using the pyruvate kinase/lactate dehydrogenase
coupled-enzyme assay. For proof of principle, the library was
screened against human R-1,3-fucosyltransferase VI (Fuc-T VI),
although there exist six different human R-1,3-fucosyltransferases.13
Three “hits” emerged from this screen, and the measurement of
the IC50 values of these three crude compounds showed that
molecule 24 had the highest affinity for Fuc-T VI (see Supporting
Information).
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10.1021/ja0302836 CCC: $25.00 © 2003 American Chemical Society

C O M M U N I C A T I O N S
The Cu(I)-catalyzed, stepwise variant10,11 of Huisgen’s classic
1,3-dipolar cycloaddition process15 seems to be the best example
of click chemistry reliability to date. It enabled a rapid synthesis
of a GDP-triazole library of 85 compounds, among which one potent
inhibitor of fucosyltransferases was identified. This method may
be widely applicable for the identification of high-affinity inhibitors
of other group-transfer targets that are of biological or medicinal
interest.
Acknowledgment. This research was supported by the NIH
(C.-H.W. and K.B.S.) and National Science Foundation (K.B.S.).
We thank William Web and Scripps Center for Mass Spectrometry
for valuable technical support with mass analysis, and Professor
W. W. Cleland and Dr. Thomas Tolbert for helpful suggestions on
the manuscript. Fuc-T III was generously provided by Dr. Nahid
Razi and The Consortium for Functional Glycomics (NIHGMS -
GM 62116).
Figure 2. Inhibition study of human R-1,3-fucosyltransferase VI. (A)
Double-reciprocal plot of GDP-fucose as the variable substrate and 24 as
the inhibitor (0, 0.4, 0.8, 1.2 µM). LacNAc concentration was held constant
at 0.5 mM. (B) Double-reciprocal plot of LacNAc as the variable substrate
and 24 as the inhibitor (0, 0.4, 1.0 µM). GDP-fucose was held constant at
10 µM.
Table 1. Inhibition Constants of 24 for Various Enzymes
enzyme
IC50 (µM)
K (nM)
i
Supporting Information Available: 1H and 13C NMR data of
compound 24 and its components (azide and GDP-alkyne); procedure
for the synthesis of azides and triazole compounds and measurements
of IC50 and Ki values (PDF). This material is available free of charge
via the Internet at http://pubs.acs.org.
R-1,3-FucT III
R-1,3-FucT V
R-1,3-FucT VI
R-1,3-GalT
â-1,4-GalT
guanylate kinase
pyruvate kinase
1.0 ( 0.2
0.9 ( 0.1
0.15 ( 0.03
N.I.
N.I.
250 ( 60
N.I.
-
270 ( 30
62 ( 3
-
-
-
-
References
(1) (a) Staudacher, E. Trends Glycosci. Glycotechnol. 1996, 8, 391-408. (b)
Sears, P.; Wong, C.-H. Cell Mol. Life Sci. 1998, 54, 223-252.
(2) Compain, P.; Martin, O. R. Bioorg. Med. Chem. 2001, 9, 3077-3092.
(3) Qian, X.; Palcic, M. M. In Carbohydrates in Chemistry and Biology; Ernst,
B., Ed.; Wiley-VCH Verlag GmbH: Weinheim, Germany, 2000; Vol. 3,
pp 293-312.
a
N.I. No Inhibition observed at 600 µM.
Steady-state kinetic evaluation of purified 24 showed that it is a
competitive inhibitor against GDP-fucose with Ki (comp) ) 62 nM
(Figure 2A, Table 1), which would make this compound the first
nanomolar and most potent inhibitor of Fuc-Ts. The inhibition
constant of 24 represents an 800-fold improvement over GDP-
alkyne (Ki ) 47 µM). Compound 24 is a noncompetitive inhibitor
against the acceptor molecule, N-acetyllactosamine (LacNAc), with
Ki (noncomp) ) 221 ( 8 nM (Figure 2B), and is a mixed-type inhibitor
against LacNAc-â-biphenyl, with Ki-slope ) 120 ( 30 nM and
Ki-intercept ) 1000 ( 400 nM.14
The inhibition property of 24 was also tested against other
glycosyltransferases and nucleotide binding enzymes (Table 1). As
is evident from the table, 24 is a potent and highly selective inhibitor
of Fuc-T VI, the enzyme from which the library was screened
against. The molecule exhibited lower inhibition properties against
other Fuc-Ts, and no inhibition was observed against two galac-
tosyltransferases. Weak inhibition was observed for guanylate
kinase, and no inhibition was observed against the catalytically
promiscuous pyruvate kinase.
(4) Mitchell, M. L.; Tian, F.; Lee, L. V.; Wong, C.-H. Angew. Chem., Int.
Ed. 2002, 41, 3041-3044.
(5) Murray, B. W.; Takayma, S.; Schultz, J.; and Wong, C.-H. Biochemistry
1996, 35, 11183-11193.
(6) Miura, Y.; Kim, S.; Etchison, J. R.; Ding, Y.; Hindsgaul, O.; and Freeze,
H. Glycoconjugate J. 1999, 16, 725-730.
(7) de Vries, T.; Palcic, M. P.; Schoenmakers, P. S.; van den Eijnden, D. H.;
and Joziasse, D. H. Glycobiology, 1997, 7, 921-927.
(8) Kolb, H. C.; Finn, M. G.; and Sharpless, K. B. Angew. Chem., Int. Ed.
2001, 40, 2004-2021.
(9) Lewis, W. G.; Green, L. G.; Grynszpan, F.; Radic, Z.; Carlier, P. R.;
Taylor, P.; Finn, M. G.; and Sharpless, K. B. Angew. Chem., Int. Ed.
2002, 41, 1053-1057.
(10) (a) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew.
Chem., Int. Ed. 2002, 41, 2596-2599. (b) Tornφe, C. W.; Christensen,
C.; Meldal, M. J. Org. Chem. 2002, 67, 3057-3064.
(11) Fazio, F.; Bryan, M. C.; Blixt, O.; Paulson, J. C.; Wong, C.-H. J. Am.
Chem. Soc. 2002, 124, 14397-14402.
(12) Mong, K.-K. T.; Lee, V. L.; Brown, J. R.; Esko, J. D.; Wong, C. H. Several
LacNAc derivatives with different aglycons were prepared and tested,
and it was found that the aglycon binding site for Fuc-T VI is hydrophobic.
(13) de Vries, T.; Knegtel, R. M. A.; Holmes, E. H.; Macher, B. A.
Glycobiology 2001, 11, 119R-128R.
(14) The inhibition patterns as displayed by FucT VI do not permit conclusive
determination of inhibitor type (mono- or bisubstrate analogue). For an
ordered mechanism, which FucT VI most likely follows,5 either mono-
or bisubstrate analogue would produce the observed competitive (versus
GDP-fucose) and noncompetitive (versus LacNAc or LacNAc-biphenyl)
patterns.
Potent inhibitors of fucosyltransferases, and glycosyltransferases
in general, have been elusive due to the aforementioned difficulties
surrounding the family of glycosyltransfer reactions. However, the
problems of weak substrate affinity and low catalytic proficiency
of the enzyme may be offset by recruiting additional binding
features, such as hydrophobic interactions in this case.
(15) Huisgen, R. In 1,3-Dipolar Cycloaddition Chemistry; Padwa, A., Ed.;
Wiley: New York, 1984; Chapter 1, pp 1-176.
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