Novel heparin/heparan sulfate mimics as inhibitors of HGF/SF-induced MET activation

Article abstract of DOI:10.1016/j.bmcl.2007.08.074

The synthesis of simple, non-sugar glycosaminoglycan (GAG) mimics has been achieved and the analogues evaluated for their ability to inhibit the activation of the MET receptor by hepatocyte growth factor/scatter factor (HGF/SF).

Full text of DOI:10.1016/j.bmcl.2007.08.074

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 17 (2007) 6321–6325
Novel heparin/heparan sulfate mimics as inhibitors
of HGF/SF-induced MET activation
Eun-Ang Raiber,a James A. Wilkinson,a,* Fabrizio Manetti,c Maurizio Botta,c
Jon Deakin,b John Gallagher,b Malcolm Lyonb and Sylvie W. Duckia,*
aCentre for Molecular Drug Design, Cockcroft Building, University of Salford, Salford M5 4WT, UK
bCancer Research UK Glyco-Oncology Group, Paterson Institute for Cancer Research, University of Manchester,
Manchester M20 4BX, UK
cDipartimento Farmaco Chimico Tecnologico, Universita degli Studi di Siena, 53100 Siena, Italy
Received 14 May 2007; revised 30 August 2007; accepted 31 August 2007
Available online 4 September 2007
Abstract—The synthesis of simple, non-sugar glycosaminoglycan (GAG) mimics has been achieved and the analogues evaluated for
their ability to inhibit the activation of the MET receptor by hepatocyte growth factor/scatter factor (HGF/SF).
ꢀ 2007 Elsevier Ltd. All rights reserved.
Cancer progression involves the proliferation and inva-
sion of cancer cells, the stimulation of tumor angiogen-
esis, and the metastasis of tumor cells through the
vasculature. Key factors in these cellular processes in-
volve growth factors such as hepatocyte growth factor/
scatter factor (HGF/SF).1 HGF/SF activates a single,
unique tyrosine kinase receptor, MET, which is the
product of the c-met proto-oncogene.2,3 There is strong
evidence that over-expression or dysregulation of the
HGF/SF-MET system in a large variety of tumors is a
major contributor to their growth, invasion, and metas-
tasis. Moreover, it has been demonstrated that efficient
activation of the MET receptor requires HGF/SF
engagement with a GAG co-receptor, which can be hep-
arin (H), heparan sulfate (HS), or dermatan sulfate
(DS),4 and probably subsequent formation of a signal-
ing ternary complex.5 Therefore disrupting productive
HGF/SF-MET interactions, via inhibition of the
co-receptor role of GAGs, may be useful in preventing
tumor invasion and metastasis.
Previous studies of H/HS interaction with HGF/SF have
shown that a tetrasaccharide is the minimal active spe-
cies.6,7 There is also evidence that trisaccharides may
be capable of interacting with HGF/SF.8 Previous stud-
ies suggested that neither N-sulfates9 nor 2-O-sulfates10
were specifically required in H/HS binding, but that hex-
osamine O-sulfation might be most important.6 How-
ever, more recent work has indicated that overall
sulfate density is more critical than strict sulfate posi-
tioning.11 Specific enzymatic and chemical scission of
H/HS, together with chemical N-desulfation, suggested
that the hexosamine 6-O-sulfate might be the most crit-
ical sulfate group in H/HS for HGF/SF recognition.
A crystal structure (PDB:1GMN) of heparin bound to
NK1, a truncated form of HGF/SF, has recently pro-
vided structural insight into the function of heparin.12
Heparin is positioned on a positively charged surface
on the N domain, possibly extending to the K1 Kringle
domain. The GAG-binding properties of individual do-
mains of HGF/SF have confirmed the major binding site
in the N-domain, and also pointed to a secondary site in
K1.13 Key interactions are mainly observed with a tetra-
saccharide segment and involve both hydrogen and ionic
bonds with sulfate and carboxylic groups. Given the po-
tential significance of therapies based on control of
GAG–protein interactions, the development of structur-
ally simpler GAG mimics is an attractive area for
investigation.
Keywords: Heparin/heparan sulfate (H/HS); Hepatocyte growth factor/
scatter factor (HGF/SF); Tyrosine kinase receptor MET; Glycosami-
noglycan (GAG); Non-carbohydrate mimic.
*
Corresponding authors. Tel.: +44 161 295 4046; fax: +44 161 295
5111 (J.A.W.); tel.: +44 161 295 4701; fax: +44 161 295 5111
(S.W.D.); e-mail addresses: j.a.wilkinson@salford.ac.uk; s.ducki@
salford.ac.uk
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.08.074

6322
E.-A. Raiber et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6321–6325
The successful use of non-carbohydrate compounds
such as suramin and its analogues for inhibitory binding
to the fibroblast growth factor (FGF) suggests that the
synthesis of aromatic, non-sugar compounds as poten-
tial inhibitors is an attractive alternative to relatively
complex sugar synthesis.14 Here we present the synthesis
and biological evaluation of a series of simple, non-su-
gar mimics of the trisaccharide 1 as inhibitors of growth
factor HGF/SF-activation of receptor (MET).
migration, and differentiation. One important compo-
nent of this cascade is the activation by di-phosphoryla-
tion of the ERK mitogen activated protein kinase
(MAPK) which then translocates to the nucleus. Phos-
phorylation of ERK can be monitored by Western blot-
ting as a downstream indicator of receptor activation by
growth factor.
The five analogues were evaluated for their ability to in-
hibit ERK phosphorylation elicited by two growth fac-
tors: HGF/SF and FGF-2 (Fig. 2).
The design of the first generation of mimics 2 was based
on the trisaccharide structure with terminal hexosamines
and an internal uronate (Fig. 1). We envisaged an aro-
matic skeleton to mimic the central carbohydrate resi-
due, bearing diametrically opposite aliphatic chains
with variable patterns of sulfate groups that would be
able to occupy similar spatial orientations as the sulfates
in the model trisaccharide.
Two compounds (7 and 12) were found to have weak
but differential inhibitory activities. Both compounds
were able to completely inhibit HGF/SF activity
(Fig. 2a) but not FGF-2 activity (Fig. 2b) at concentra-
tions of 100 lg/mL. At lower concentrations neither
compound 7 (Fig. 3a) nor 12 (Fig. 3b) showed any
inhibitory activity toward ERK phosphorylation in-
duced by HGF/SF.
The synthesis of heparin mimics was straightforward. It
was designed to introduce different degrees of sulfation,
demonstrated to be an important feature for binding
affinity (Scheme 1). Briefly, methylation and allylation
(steps i, ii or (v) a) of 2,5-dihydroxybenzoic acid 3 led
to mono- and di-allyl methyl esters. The Sharpless asym-
metric dihydroxylation.15,16 (AD steps (iii) a and (iv) a)
was used to enantioselectively prepare 1,2-diols which
were sulfated17 (steps (iii) b and (iv) b) to afford ana-
logues 6 and 7. Mono-allyl ester was subjected to reac-
tion with 4-bromobutyl acetate (step (v) b) to afford
compound 8, followed by AD and sulfation (step vi)
to give analogue 9. Finally analogues 11 and 12 were ob-
tained using similar sequences (steps viii and ix) from
compound 10.
The discrimination in the inhibition of different GAG-
dependent growth factors demonstrates the potential
for selective clinical targeting via antagonism of the
co-receptor mechanism.
Madin–Darby canine kidney (MDCK) cells are com-
monly used for assaying HGF/SF activity in vitro. These
cells will actively migrate across a porous Transwell
membrane in a chemotactic response to HGF/SF.18
Compounds 6, 7, 9, 11, and 12 were evaluated in the
migration assay (Fig. 4). Compound 7 was able to inhi-
bit HGF/SF-induced cell migration by 57% while com-
pound 12 inhibited it by 70%.
The inhibitory properties of the heparin mimics 6, 7, 9,
11, and 12 were first determined in wild type Chinese
hamster ovary (CHO) cells, which naturally express
both MET and FGF receptors, as well as GAG co-
receptors. FGF activity is also dependent upon H/HS
co-receptors. Receptors are activated upon productive
binding of the respective growth factors leading to phos-
phorylation of their intrinsic tyrosine kinase domains.
This event initiates an intracellular signaling cascade
resulting in cellular responses such as proliferation,
Although compounds 7 and 12 showed only a micromo-
lar inhibitory activity toward HGF/SF, they are the first
sugar mimetic compounds described to demonstrate sig-
nificant inhibition of both HGF/SF-induced ERK acti-
vation and HGF/SF-induced migration in MDCK cells.
A computational study was undertaken to predict possi-
ble binding modes for heparin mimic 12. The model was
built based on the X-ray crystal structures of NK1
˚
(PDB: 1BHT, 2.0 A resolution) and NK1-heparin
˚
(PDB: 1GMN, 3.0 A resolution). In order to check that
the selected blind docking process was adequate for find-
ing the correct binding mode of compound 12, the hep-
arin tetradecasaccharide was extracted from the original
structure 1GMN and docked into the better resolved
1BHT using AUTODOCK 3.0 (100 runs).19 The docked
OSO3
O
OSO3
O
O2C
O
O
O
O
O
˚
structures were clustered at 1.0 A rmsd. The most pop-
ulated cluster (62 conformations) contained the heparin
conformation with the lowest docking energy
(E = À589.59 kJ molÀ1). The binding mode of the low-
est energy complex was submitted to energy minimiza-
tion and was compared to the crystal structure. The
docking study results in a binding mode for heparin that
is almost identical to that found in the original X-ray
HO
NH
SO3
O
HO
NH
SO3
SO3
1
OSO3
O2C
O
O
SPACER
˚
structure (rms 1.0 A).
O
NH
2
SO3 SO3
Compound 12 was docked using the methodology de-
scribed above (250 runs).20 The final complexes were
Figure 1. Design of non-carbohydrate heparin mimics.

E.-A. Raiber et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6321–6325
6323
OSO3H
COOMe
HO3SO
O
COOH
COOMe
iii
COOMe
O
OSO3H
OSO3H
HO
HO
O
i
ii
6
iv
OH
OH
O
OSO3H
O
COOH
3
5
4
HO3SO
v
O
OSO3H
OSO3H
O
COOMe
7
O
COOMe
vi
O
O
O
O
O
OSO3H
OSO3H
O
8
9
vii
COOH
O
HO3SO
HO3SO
COOH
O
O
OSO3H
OSO3H
viii
O
HO
11
COOH
10
ix
O
O
OSO3H
OSO3H
12
Scheme 1. Reagents and conditions: (i) MeOH, H2SO4, reflux, 24 h, 95%; (ii) NaH (3 eq), AllylBr (3 equiv), t-Bu4NI (0.2 equiv), DMF, rt, 12 h, 92%;
(iii) a—AD mix a, MeSO2NH2 (1 equiv), t-BuOH/H2O, 0 ꢁC, 12 h, 61%; b—SO3ÆNMe3, DMF, 40 ꢁC, 12 h, 55%; (iv) a—KOH (2 equiv), water/
ethanol, 6 h, 60 ꢁC, 70%; b—AD mix b, MeSO2NH2 (1 equiv), t-BuOH/H2O, 12 h, 0 ꢁC, 58%; c—SO3ÆNMe3, DMF, 40 ꢁC, 12 h, 42%; (v) a—K2CO3
(1.5 equiv), AllylBr (1.5 equiv), n-Bu4NI (0.2 equiv), acetone, rt, 12 h, 68%; b—NaH, 60% (1.5 equiv), Br(CH2)4OAc (1.5 equiv), n-Bu4NI (0.2 equiv),
DMF, 12 h, 71%; (vi) a—AD mix a, MeSO2NH2 (1 equiv), t-BuOH/H2O, 0 ꢁC, 12 h, 58%; b—SO3ÆNMe3, DMF, 40 ꢁC, 12 h, 73%; (vii) KOH (2
equiv), water/ethanol, rt, 60 ꢁC, 6 h, 70%; (viii) a—AD mix a, MeSO2NH2 (1 equiv), t-BuOH/H2O, 0 ꢁC, 12 h, 86%; b—SO3ÆNMe3, DMF, 40 ꢁC,
12 h, 83%; (ix) a—AD mix b, MeSO2NH2 (1 equiv), t-BuOH/H2O, 12 h, 0 ꢁC, 80%; b—SO3ÆNMe3, DMF, 40 ꢁC, 12 h, 79%.
a) HGF/SF induced ERK activation
a) Concentration-dependent ERK activation (compound 7)
100 10 100 10
-ve +ve µg/ml µg/ml µg/ml µg/ml µg/ml
1 1
6
7
9
12
1
-ve + ve
ppERK
ppERK
b) FGF-2 induced ERK activation
11
6
7
9
1 2
-ve + ve
b) Concentration-dependent ERK activation (compound 12)
100 10 100 10
-ve +ve µg/ml µg/ml µg/ml µg/ml µg/ml
1
ppERK
Figure 2. Effect of heparin mimics 6, 7, 9, 11, and 12 on growth factor-
induced ERK activation. CHO cells were treated for 20 min with either
(a) HGF/SF or (b) FGF-2 and candidate analogues (100 lg/mL). Cell
extracts resolved by SDS–PAGE were probed for activated ERK by
Western blotting.
ppERK
Figure 3. Concentration-dependent effect of compounds 7 and 12 on
HGF/SF-induced ERK activation.
and compound 12 could be identified (Fig. 5). Com-
pound 12 interacts with residues Arg35, Arg76, Arg73,
Lys78, Lys60, and Lys62 through hydrogen/ionic inter-
actions. Residues Arg73, Lys60, and Lys62 are known
to play an important role in heparin-HGF/SF binding.
optimized by energy minimization using Macromodel.21
˚
Finally, the docked structures were clustered at 4.0 A
rmsd. Thirty-eight distinct conformational clusters were
found. The most populated cluster (73 conformations)
contained the conformation with the lowest docking en-
ergy (E = À14.67 kcal molÀ1).
In summary, a series of structurally-related heparin
mimics were synthesized in 4–6 steps. These mimics bear
sulfate groups responsible for the binding of the H/HS
to the growth factor. They were evaluated for their
After minimization of the docked complex, amino acid
residues involved in the interaction between the protein

6324
E.-A. Raiber et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6321–6325
acknowledge EPSRC for provision of accurate mass
spectral data.
References and notes
1. Galzie, Z.; Kinsella, A. R.; Smith, J. A. Biochem. Cell Biol.
1997, 75, 669.
2. Bottaro, D. P.; Rubin, J. S.; Faletto, D. L.; Chan, A. M.-
L.; Kmiecik, T. E.; Vande Woude, G. F.; Aaronson, S. A.
Science 1991, 251, 802.
3. Naldini, L.; Weidner, K. M.; Vigna, E.; Gaudino, G.;
Bardelli, A.; Ponzetto Narsimhan, C. R. P.; Hartmann,
G.; Zarnegar, R.; Michalopoulos, G. K.; Birchmeier, W.;
Comoglio, P. M. EMBO J. 1991, 10, 2867.
4. Lyon, M.; Deakin, J. A.; Rahmoune, H.; Fernig, D. G.;
Nakamura, T.; Gallagher, J. T. J. Biol. Chem. 1998, 273,
271.
5. Lyon, M.; Deakin, J. A.; Gallagher, J. T. J. Biol. Chem.
2002, 277, 1040.
6. Delehedde, M.; Lyon, M.; Vidyasagar, R.; McDonnell, T.
J.; Fernig, D. G. J. Biol. Chem. 2002, 277, 12456.
7. Lyon, M.; Deakin, J. A.; Lietha, D.; Gherardi, E.;
Gallagher, J. T. J. Biol. Chem. 2004, 279, 43560.
8. Lyon, M. unpublished data.
Figure 4. Effect of analogues 6, 7, 9, 11, and 12 on the Transwell
migration of MDCK cells. MDCK cells (2 · 105 cells/well) were seeded
onto Transwell membrane (pore size 8 lm) and incubated for 4 h at
37 ꢁC in the absence (À) or presence (+) of HGF/SF and heparin
mimics (100 lg/mL). Migrated cells were stained with Crystal Violet
and counted, to give a mean cell count.
9. Lyon, M.; Deakin, J. A.; Mizuno, K.; Nakamura, T.;
Gallagher, J. T. J. Biol. Chem. 1994, 269, 11216.
10. Merry, C. L. R.; Bullock, S. L.; Swan, D. C.; Backen, A.
C.; Lyon, M.; Beddington, R. S. P.; Wilson, V. A.;
Gallagher, J. T. J. Biol. Chem. 2001, 276, 35429.
11. Catlow, K. R.; Deakin, J. A.; Wei, Z.; Delehedde, M.;
Fernig, D. G.; Gherardi, E.; Gallagher, J. T.; Pavao, M. S.
G.; Lyon, M. J. Biol. Chem. 2007, submitted for
publication.
12. Lietha, D.; Chirgadze, D. Y.; Mulloy, B.; Blundell, T. L.;
Gherardi, E. EMBO J. 2001, 20, 5543.
13. Holmes, O.; Pillozzi, S.; Deakin, J. A.; Carafoli, F.; Kemp,
L.; Butler, P. J. G.; Lyon, M.; Gherardi, E. J. Mol. Biol.
2007, 367, 395.
14. Manetti, F.; Cappello, V.; Botta, M.; Corelli, F.; Mongelli,
N.; Biasoli, G.; Borgia, A. L.; Ciomei, M. Bioorg. Med.
Chem. 1998, 6, 947.
15. Mehltretter, G. M.; Dobler, C.; Sundermeier, U.; Beller,
M. Tetrahedron Lett. 2000, 41, 8083.
Figure 5. Proposed binding mode for compound 12 (orange) with
HGF/SF (gray).
16. General procedure for Sharpless AD: AD a mix (5.54 g)
was dissolved in a mixture of t-BuOH/water (20 mL/
20 mL) and cooled down to 0 ꢁC. The alkene (2.0 mmol)
and methansulfonamide (2.0 mmol) were added and the
reaction mixture was stirred at 0 ꢁC for 12 h. The
reaction was quenched with sodium sulfite (5 g) and the
solvent was removed in vacuo. Ethanol (15 mL) was
added to the crude mixture and the mixture was heated
under reflux for 1 h. The salt was filtered off and the
crude compound purified by flash column chromatogra-
phy on silica.
17. General procedure for sulfation: The alcohol (0.3 mmol)
was dissolved in N,N-dimethylformamide (3.8 mL).
Sulfur trioxide-trimethylamine complex (1.68 mmol)
was added and the reaction mixture was stirred at
40 ꢁC for 12 h. The solvent was removed in vacuo and
the crude product was purified by flash column
chromatography.
ability to inhibit HGF/SF- or FGF-2-mediated activa-
tion of ERK MAPK in wild type CHO cells. Two com-
pounds 7 and 12 were found to have good, selective
inhibitory activity. Both compounds were also able to
significantly inhibit HGF/SF-induced cell migration in
MDCK cells. Simulated docking experiments suggest
that compound 12 may compete for binding to part of
the H/HS binding site in HGF/SF. These results are
promising and demonstrate the potential for these aro-
matic sugar mimetics to selectively inhibit HGF/SF.
We now intend to design a second generation of H/HS
mimics based upon our preliminary docking model.
Acknowledgments
18. Stoker, M. J. Cell. Physiol. 1989, 139, 565.
19. Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.;
Hart, W. E.; Belew, R. K.; Olson, A. J. J. Comput. Chem.
1998, 19, 1639.
20. Autodock parameters: Translation, Quaternion and Tor-
sion reduction factor 1/cycle; No. of top inividudals that
The Biomedical Science Research Institute at Salford
and Cancer Research, UK, have generously supported
our work throughout this time. We would like to thank
Boehringer Ingelheim for a travel fund for E-AR. We

E.-A. Raiber et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6321–6325
6325
automatically survive 1; Rate of gene mutation 0.02; Rate
of crossover 0.8; No. of generations for picking worst
individual 10; Mean of Cauchy distribution for gene
mutation 0; Variance of Cauchy distribution for gene
mutation 1; No. of iterations of Solis and Wets local
search 300; No. of consecutive successes before changing p
4; No. of consecutive failures before changing p 4; Size of
local search space to sample 1; Lower bound on p 0.01;
Probability of performing local search on an individual
0.06; Total number of energy evaluations 10 million;
Populations in the generic algorithm 250.
21. Mohamadi, F.; Richards, N. G. J.; Guida, W. C.; Liskamp,
R.; Lipton, M.; Caufield, C.; Chang, G.; Hendrickson, T.;
Still, W. C. J. Comput. Chem. 1990, 11, 440.