Dendrimer Heparan Sulfate Glycomimetics: Potent Heparanase Inhibitors for Anticancer Therapy

Article abstract of DOI:10.1021/acschembio.8b00909

Heparanase is a mammalian endoglycosidase that cleaves heparan sulfate (HS) polysaccharides and contributes to remodelling of the extracellular matrix and regulation of HS-binding protein bioavailabilities. Heparanase is upregulated in malignant cancers and inflammation, aiding cell migration and the release of signaling molecules. It is established as a highly druggable extracellular target for anticancer therapy, but current compounds have limitations, because of cost, production complexity, or off-target effects. Here, we report the synthesis of a novel, targeted library of single-entity glycomimetic clusters capped with simple sulfated saccharides. Several dendrimer HS glycomimetics display low nM IC₅₀ potency for heparanase inhibition equivalent to comparator compounds in clinical development, and potently inhibit metastasis and growth of human myeloma tumor cells in a mouse xenograft model. Importantly, they lack anticoagulant activity and cytotoxicity, and also inhibit angiogenesis. They provide a new candidate class for anticancer and wider therapeutic applications, which could benefit from targeted heparanase inhibition.

Full text of DOI:10.1021/acschembio.8b00909

Subscriber access provided by Kaohsiung Medical University
Letter
Dendrimer Heparan Sulfate Glycomimetics: Potent
Heparanase Inhibitors for Anticancer Therapy
Olga V. Zubkova, Yassir A. Ahmed, Scott E. Guimond, Sophia-Louise Noble,
John Holmes Miller, Raymond Alexander Alfred Smith, Victor Nurcombe,
Peter C. Tyler, Marina Weissmann, Israel Vlodavsky, and Jeremy E. Turnbull
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.8b00909 • Publication Date (Web): 27 Nov 2018
Downloaded from http://pubs.acs.org on November 28, 2018
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 8
ACS Chemical Biology
1
2
3
4
5
6
7
8
9
Dendrimer Heparan Sulfate Glycomimetics: Potent Heparanase
Inhibitors for Anticancer Therapy
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Olga V. Zubkova,*,§ Yassir A. Ahmed,† Scott E. Guimond,† Sophia-Louise Noble,‡ John Holmes
Miller,‡ Raymond Alexander Alfred Smith,& Victor Nurcombe,& Peter C. Tyler,§ Marina
Weissmann,# Israel Vlodavsky,# and Jeremy E. Turnbull†
§
The Ferrier Research Institute, Victoria University of Wellington, Gracefield Research Centre, Lower Hutt, New
Zealand
†
Institute of Integrative Biology, University of Liverpool, Liverpool L69 3BX, United Kingdom
School of Biological Sciences, Victoria University of Wellington, Kelburn, Wellington, New Zealand
‡
&
Glycotherapeutics Group (VNSC), Institute of Medical Biology, A*STAR, Singapore
#
Cancer and Vascular Biology Research Center, Rappaport Faculty of Medicine, Technion-Israel Institute of
Technology, Haifa, Israel
ABSTRACT:
Heparanase is a mammalian endoglycosidase that cleaves heparan sulfate (HS) polysaccharides and
contributes to remodelling of the extracellular matrix and regulation of HS-binding protein bioavailabilities. Heparanase is
upregulated in malignant cancers and inflammation, aiding cell migration and the release of signalling molecules. It is
established as a highly druggable extracellular target for anticancer therapy, but current compounds have limitations due
to cost, production complexity or off-target effects. Here we report the synthesis of a novel, targeted library of single-entity
glycomimetic clusters capped with simple sulfated saccharides. Several dendrimer HS glycomimetics display low nM
IC50 potency for heparanase inhibition equivalent to comparator compounds in clinical development, and potently inhibit
metastasis and growth of human myeloma tumor cells in a mouse xenograft model. Importantly, they lack anticoagulant
activity and cytotoxicity, and also inhibit angiogenesis. They provide a new candidate class for anti-cancer and wider
therapeutic applications which could benefit from targeted heparanase inhibition.
Heparan sulfate (HS) is a highly sulfated glycosaminoglycan with a variety of critical functions in cell signalling and
regulation1. HS acts as an extracellular storage depot of growth factors, angiogenic proteins, and chemokines, and also
serves to regulate ligand-receptor interactions at the cell surface2-3. Heparanase is the only known endo-β-D-
glucuronidase capable of cleaving HS side chains of HS proteoglycans (HSPGs) into shorter, active fragments4-5. Cleavage
of HS by heparanase facilitates structural alterations of the extracellular matrix (ECM) and contributes to releasing
various sequestered HS-binding proteins6. Heparanase is upregulated in malignant cancers, and its overexpression
correlates with angiogenesis, increased tumor size, enhanced metastasis, and poor prognosis7-8. The upregulation of
heparanase in malignant cancer also correlates closely with increases in new blood vessels around tumors, providing a ready
pathway for cancer cell entry into the circulation9. This makes heparanase a promising extracellular target for new anti-
tumor treatments.
While HS oligosaccharides can mimic or interfere with biological systems, their exploitation has been hindered by the
complexity of their synthesis. Recently we developed a novel synthetic approach to more accessible polyvalent HS-mimetic
constructs with potential advantages as therapeutics10. We employed the clustering effect of dendrimers in which weak
interactions of small sugar fragments are significantly enhanced by appending multiple copies of specific sugars on defined
chemical scaffolds11. Our targeted libraries of single-entity HS oligosaccharides and glycomimetic clusters have already been
demonstrated to mimic longer natural HS molecules that have a role in Alzheimer’s disease12-14. The ability of the target
compounds to inhibit the BACE-1 protease was investigated using fluorescence resonance energy transfer (FRET) peptide
cleavage assays. All of the glycomimetic clusters inhibited BACE-1 with IC50 values in the micromolar to low nanomolar
range and showed a lack of off-target anticoagulant activity.
We reasoned that our dendritic cores and associated methodology would provide a novel template for the polyvalent
presentation of other HS-mimicking saccharides for other indications. Here we extended our research into anticancer
applications and screened a library of HS-glycomimetics for their ability to inhibit heparanase.
ACS Paragon Plus Environment

ACS Chemical Biology
Page 2 of 8
1
2
3
4
-
O3SO
O2C
A.
H
N
O
O
O
-
-
-
-
B.
O
-
(CH2)6
OSO3
OSO3
(CH2)7
O3SO
HO
O2C
N
C
OH
OH
(CH2)6 NH CO L1 CH2-O-CH2
H
O
O
O
-
OSO3
O
O
O
O
C
-
O
CH2
C
O
OSO3
OH
OH
-O3SHN
-
OSO3
1
-O3SO
O
O
-
-O3SHN
OSO3
OSO3
OSO3
5
4
4
n = 0 - 1
6
7
8
9
L1 = a, CH2; b, CH2CONH(CH2)7; c, CH2CO-NH(CH2)7-CONH-((CH2)2O)3-(CH2)2
26 - 31
OAc
O
OAc
O
OR1
OR1
O
C.
OAc
OAc
OAc
O
(CH2)6
Z
O
OR1
OR1
D.
O
AcO
O
O
O
C
O
C
OAc
OAc
n = 0 - 1
R1O
O
N
O
OR1
O
O
O
N
O
O
O N
O
N O
OR1
O
O
CHN
N C
H
O
O
O
Ref.10
O
O
O
O
O
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
O
O
O
O
n = 0 - 1
O
O
2: n=0; 3: n=1
O
O
i
O
H
O
O N
R1 = Ac: 5: n=0, Z=Cl; 6: n=1, Z=Cl
R1 = Ac: 7, n=0, Z=N3; 8, n=1, Z=N3
R1 = H: 9, n=0, Z=N3; 10, n=1, Z=N3
N
C
N
+
C N
C
N O
O
C
O N
O
H
O
ii
O
13
Ref.10
HO
O
O
O
14
Ref.10
Cl
O
iii
iv
4
v
O
O
O
O
O
CO2Bn +
R1 = H: 11, n=0, Z=NH2; 12, n=1, Z=NH2
H2N
H2N
O
CO2H
vi
16
15
O
R2
H
O
O
O
O
NH
O
O
R2
O
C
O
O
O
N
O
N C
NH
O
O
11 + 13
12 + 13
O
O
H
O
O
E.
O
O
O
O
O
H
R2
H
O
N
N
O
O
C
N
C N
O
O
R2
O
R3O
O
OR3
O
H
H
O
R3O
R3O
O
R2 = Bn: 17
R2 = H: 18
R2 = Su: 19
ix
vii
viii
O
OR3
OR3
OR3
OR3
O
OR3
R3O
O
R3O
O
O
O
O
R3O
OR3
O
C
H2C
OR3
HN
OR3
R3O
R3O
OR3
O
C
O
O
O
R3O
CH2
O
O
O
O
H
H
H
H
H
OR3
n = 0
O
R3O
C
N
C
NH
C
11 + 14
12 + 14
C
O
- 1
O
OR3
O
OR3
R3O
O
OR3
R3O
R3O
O
O
OR3
O
R3O
O
R3O
OR3
O
ix
O
O
L1 = CH2
H
N
OR3
OR3
O
O
OR3
R3O
R3O
C
R3O
OR3
NH
O
OR3
OR3
R3O
R3 = H: 20: n=0, L1 = CH2; 23: n=1, L1 = CH2
R3 = SO3-: 26: n=0, L1 = CH2; 29: n=1, L1 = CH2
O
O
OR3
H
N
O
x
HN
R3O
O
R3O
O
C
OR3
O
R3O
O
O
O
C
O
O
CH2
O
R3O
O
NH
OR3
R3O
OR3
R3O
C
O
OR3
N
n = 0 - 1
O
R3O
R3O
O
HN
NH
H
O
OR3
O
OR3
O
L1 = CH2CONH(CH2)7
R3 = H: 21: n=0, L1 = CH2CONH(CH2)7; 24: n=1, L1 = CH2CONH(CH2)7
R3 = SO3-: 27: n=0, L1 = CH2CONH(CH2)7; 30: n=1, L1 = CH2CONH(CH2)7
O
x
R3O
R3O
O
OR3
O
11 + 19
R3O
12 + 19
O
O
R3O
O
O
H
OR3
R3O
O
C
O
O
N
O
O
OR3
C
O
R3O
OR3
N
O
ix
R3O
R3O
H
NH
O
C
O
O
OR3
HN
O
H
O
O
N
OR3
O
C
O
O
O
R3O
O
R3O
C
O
OR3
O
R3O
O
n = 0 - 1
OR3
NH
O
R3O
O
R3O
O
C
O
O
O
OR3
n = 0 - 1
C
O
N
R3O
OR3
N
O
O
R3O
NH
H
H
O
C
O
O
OR3
O
R3O
HN
O
O
OR3
O
L1 = CH2CO-NH(CH2)7-CONH-((CH2)2O)3-(CH2)2
O
R3 = H: 22: n=0, L1 = CH2CO-NH(CH2)7-CONH-((CH2)2O)3-(CH2)2; 25: n=1, L1 = CH2CO-NH(CH2)7-CONH-((CH2)2O)3-(CH2)2
R3 = SO3-: 28: n=0, L1 = CH2CO-NH(CH2)7-CONH-((CH2)2O)3-(CH2)2; 31: n=1, L1 = CH2CO-NH(CH2)7-CONH-((CH2)2O)3-(CH2)2
x
Scheme 1. Synthesis of glycomimetics. (A, B) Synthetic targets. (C) Preparation of saccharide fragments: i, BF3.OEt2 , DCM,
RT; ii, NaN3, DMF, 80 ºC; iii, NaOMe, MeOH, RT; iv, Ra-Ni, H2, aq. MeOH, RT. (D) Preparation of tetramer dendritic cores:
v, BnOH, SOCl2, 0 ºC to 100 ºC; vi, Et3N, DMF, RT; vii, H2, Pd(OH)2/C, aq. MeOH, RT; viii, NHS, EDC, DMF, DIPEA, RT. (E)
Preparation of HS glycomimetics: ix, Et3N, DMF, RT; x, SO3·NMe3, DMF, 60 ºC.
ACS Paragon Plus Environment

Page 3 of 8
ACS Chemical Biology
1
2
3
A.
B.
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 1. Heparanase inhibitory activity of selected compounds. Panel A: Initial in vitro screen of heparanase inhibitory
activity of selected compounds at 1 µM concentration in a Fondiparinux substrate assay using 100 nM heparanase. Panel
B: dose response curves for heparanase inhibition by selected compounds in the Fondiparinux assay using 10 nM
heparanase. IC50 values were calculated from the curves (see Methods: PG545, 8 nM; 29, 11 nM; 30, 23 nM; 31, 116 nM).
Our approach was to alter the ECM of tumor cells and their detachment prior to metastasis by preventing heparanase
action and thereby altering the oxygen, nutrient and growth factor supply to the tumor. Standard chemotherapeutic
anticancer drugs kill rapidly dividing cancer cells but also affect normal cell proliferation, such as occurs in the bone
marrow and gut, leading to toxic side effects. In contrast, normal cells express extracellular heparanase at very low
levels, and heparanase knock-out animals exhibit no obvious deficits. These characteristics imply that inhibition of
heparanase will cause minimal side effects in cancer patients.
Initially we tested the available N-acetylated HS mimetics developed for BACE1 inhibition10; but, these did not inhibit
heparanase with any significant potency. However, one of the N-sulfated analogs (compound 1, Scheme 1, Panel A) did
show modest inhibitory activity (60% inhibition at 1 μM concentration; Figure 1, Panel A). Thus, we developed further
analogues (26-31, Scheme 1, Panel B), reasoning that increasing the level of sulfation would lead to more potent
inhibitors. As a simplified alternative to complex HS saccharides, we synthesized a novel targeted library of single-
entity dendrimer HS glycomimetic clusters capped with simple sulfated saccharides, glucose and maltose, that
potentially mimic gluco-configured HS. We also varied the length of the dendritic core arms employing PEG linkers to
increase the solubility of the glycomimetics. Using affordable starting ingredients, we also significantly shortened the
synthesis process, thus markedly reducing manufacturing costs.
Glycosylation of peracetylated sugars15, glucose (2) and maltose (3) with 6-chloroethanol (4) in dichloromethane in the
presence of boron trifluoride etherate at room temperature gave glycosides (5) and (6). Displacement of the chloro-
group with sodium azide in DMF at 80C followed by Zemplen deacetylation afforded azido-derivatives (9) and (10).
Reduction of the azides at ambient temperature and pressure with Raney Nickel under hydrogen in aqueous methanol
furnished amino-glycosides (11) and (12) in quantitative yields (Scheme 1, Panel C). “Short-armed” tetra-N-
hydroxysuccinimide activated ester 13 and a “long-armed” tetra-N-hydroxysuccinimide activated ester 14 were prepared
as described in our earlier work10. Treatment of PEG-acid (15) with benzyl alcohol and thionyl chloride gave a benzyl
ester of PEG amino acid 16. Coupling the benzyl ester 16 to a “long-armed” core 14 led to the tetravalent dendritic core
17. Hydrogenolysis of (17) gave the tetra-acid 18, and its treatment with NHS/EDC afforded the PEGylated N-
hydroxysuccinimide ester 19 (Scheme 1, Panel D).
In our previous work, we observed complex mixtures of starting material, di-, tri- and tetra-substituted clusters, when
sulfated HS fragments were attached to the tetrameric dendritic cores. This was likely due to the electrostatic repulsion
of anionic sulfo-groups. We improved the coupling yields by first attaching non-charged fragments followed by over-
sulfation. Here we followed the same strategy (Scheme 1, Panel E).
Coupling of four equivalents of glycoside fragments 11 and 12 with tetra-N-hydroxysuccinimide active esters 13, 14 and
19 gave tetramer clusters 20-25 in 85-95% yield after chromatography. Sulfation of tetramers in dry DMF under argon
at 60C for 72 h afforded the desired O-sulfated HS mimetics 26-31 in excellent yields of 88-92% after chromatography.
All six final products 26-31 and their six precursors 20-25 were analysed by 1D and 2D NMR spectroscopy in deuterium
oxide at 500 MHz. The chemical shift data are presented in the Supporting Information (Tables S1 and S2). Chemical
shifts were assigned using the HSQC technique. 1H and 13C NMR spectra indicated that compounds 26-31 were fully
sulfated since single sets of peaks were observed with resonances shifted downfield.
The ability of the target compounds to inhibit heparanase in vitro was investigated using a Fondaparinux substrate
heparanase assay16 (see Methods in the Supporting Information), and demonstrated that HS glycomimetic clusters (26-
31) inhibited heparanase with IC50 values in the micromolar to low nanomolar range (Figure 1).
Tetramers 26-28 with sulfated monosaccharide (glucose) fragments showed only weak ability to inhibit heparanase in
an initial screen at 1 M dosage (Figure 1, Panel A), with IC50 values >> 1M; in contrast, tetramers 29-31 with larger
sulfated disaccharide (maltose) residues were the most active variants (Figure 1, Panel A). Thus at least 2 sulfated
monosaccharide sugar units was critical for activity. Dose response experiments indicated that the potency was higher
for “long-armed” PET tetramer 30, (23nM) compared to the “PEGylated long-armed” cluster with sulfated maltose, 31
ACS Paragon Plus Environment

ACS Chemical Biology
Page 4 of 8
(116 nM; Figure 1, Panel B). Interestingly, a “short-armed” tetramer cluster 29 with sulfated maltose fragments showed
the highest potency, with an IC50 value of 11 nM. These results indicate that the length of the arms is a significant factor
in determining potency; this, along with potential activity of dimer constructs will be a useful avenue to explore in
further optimisation studies. The potency of the known heparanase inhibitor, PG545, was found to be 8 nM (Figure 1,
Panel B), in very good agreement with the literature28. Thus, 29 and 30 have potency very similar to the comparator
PG545 which is in clinical development25.
1
2
3
4
5
6
7
8
9
Myeloma tumors express high levels of the HSPGs on their cell surfaces17. Heparanase is upregulated in myeloma tumor
cells, and the bone marrow plasma of some patients provides a rich source of biologically active fragments that enhance
a tumor‘s growth and metastasis18. Previous work has shown that heparanase inhibitors reduce myeloma metastasis
and tumor growth in a xenograft mouse model17,19. We tested one of our most potent in vitro inhibitors 30 in this highly
aggressive myeloma model (Figure 2, Panel A), in which cells metastasize to bone marrow with tumors growing
predominantly in the bone, closely mimicking these aspects of the human disease17. We observed remarkably reduced
tumor spread and growth in the mice treated with the inhibitor (dorsal view, 85.4% inhibition, p=0.037 and ventral
view, 88.5% inhibition, p=0.024 at 4 weeks; see Methods, Figure S2), indicative of potent heparanase inhibition.
Some cancer cells form precapillary-like tubes or cords when grown in a basement membrane gel extract, a process
called vascular mimicry20. Both glycomimetics 29 and 30 inhibited vascular mimicry in cultures of B16 melanoma cells
at concentrations of 10 and 40 μM (Figure 2, Panel B), without inhibiting cell migration (Supporting Information, Figure
S3). Vascular mimicry, a process similar to angiogenesis, generates blood vessels for vascularization of tumors and
occurs in the absence of an endothelial cell line in the cultures. Prevention of neovascularization of a tumor would
contribute to inhibition of a tumor’s growth by the HS glycomimetics.
A notable potential side effect of heparin and related compounds that can limit their wider clinical application is
anticoagulant activity. Importantly, the compounds we synthesized displayed a remarkable lack of any significant
anticoagulant activity towards Factor Xa at concentrations >100-fold or higher than their IC50 for inhibiting heparanase
(Figure 2, Panel C), indicating that they differ from some other compounds in this class such as PG54523 and
necuparanib29. Importantly, we also established that this class of compounds was not cytotoxic to BaF3 mouse
lymphoid cells at 100 μg/mL (~15-32 μM), which is ~ 100-1500 fold higher than their IC50 for inhibiting heparanase (see
Supporting Information, Figure S4).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
A further known activity of heparin is regulation of fibroblast growth factors (FGFs) which are crucial for cell growth
and can also promote angiogenesis21. We observed no activation of FGF-1 or FGF-2 signalling via FGF receptor FGFR1c
with any of the compounds on BaF3 lymphoid cell proliferation (26-31, Figure 3, Panel A) where viability is dependent
on FGF signaling in the absence of Interleukin 3 (IL-3) (see Methods).
We also examined the compounds for inhibitory properties towards FGF signaling in BaF3 cells in which cells are
partially activated by addition of 3 μg/mL heparin. Notably, none of the compounds (1, 26-31, Figure 3, Panel B)
significantly inhibited FGF1 in BaF3 cells transfected with FGFR1c. In contrast, FGF2 was inhibited by some of the
compounds, most potently by compounds 30 and 31. This is significant since inhibition of FGF2 could contribute to the
anti-angiogenic properties of these compounds that we observed (Figure 2, Panel B), providing an additional anticancer
mode of action. BaF3 cells are a mouse pre-B lymphocytic cell line dependent on IL-3 for survival22. They are devoid of
heparan sulfate and FGF receptors and do not respond to FGF in the presence or absence of heparin23. BaF3 cells
transfected with FGFR1c gain the ability to respond to FGF-1 and FGF-2 in the presence of exogenously added heparin
or heparin mimetics, and the cells can proliferate normally in the absence of Il-323 (Figure 3, Panel A). If no heparin is
present or a heparin species is not capable of supporting FGF activity, the cells do not proliferate and eventually die.
There are no therapeutically effective heparanase inhibitors currently available for use in the clinic24. Heparin, an
antithrombotic drug that also inhibits heparanase, is not clinically useful because of its strong anticoagulant
activity. However, the use of HS-related compounds as antitumor agents has been reported in the literature, resulting
in clinical trials currently underway. For example, Progen Pharmaceuticals has designed a compound called PG545 that
is produced from a very expensive tetrasaccharide maltotetraose25. PG545 treatment reduces heparanase activity in
primary tumors and is presently undergoing clinical trials26.
The heparanase inhibitors necuparanib (Momenta Pharmaceuticals)27 and a glycol-split modified heparin roneparstat
(Leadiant Biosciences)28 are produced from natural heparin by chemical modifications. These compounds display
multiple anticancer activities, including heparanase inhibition and anti-angiogenic properties. However, as they are
complex mixtures, they may face hurdles in obtaining FDA approval, as the methods required to characterize batch-to-
batch variations are very elaborate and costly. Furthermore, both PG54523 and necuparanib29 also retain significant
anticoagulant activity, and roneparstat requires high dosage levels due to short half-life27; these factors that might limit
their clinical usage.
Thus, collective data point to heparanase as a good target molecule to interfere with cancer progression, though none
of the treatments already under development are ideal. Our HS glycomimetics could provide a solution to these
drawbacks since they have distinct advantages, including a unique combination of potency comparable to PG545
(currently the most potent of the described inhibitors with an IC50 value of 12 nM)30, allied with a lack of any significant
anticoagulant activity, and also lack of cell toxicity. We have described rapid, convenient, and economical methods for
the synthesis of a range of single chemical entity sulfated glycomimetics. Further studies are underway to evaluate their
pharmacokinetics, bioavailability, and safety as a prelude to potential clinical testing.
ACS Paragon Plus Environment

Page 5 of 8
ACS Chemical Biology
1
2
A.
3
4
5
6
29
(3 M)
30
(3 M)
B16
Control
B16 Cell Angiogenesis Inhibition
7
8
9
150
B.
100
50
0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
)
)
)
)
)
)
)
)
)
)
d
e
M
M
M
M
M
M
M
M
M
M
n
t
µ
µ
µ
9
µ
µ
µ
µ
0
µ
µ
l
a
e
3
7
3
3
3
7
.
1
3
(
3
3
3
3
.
.
.
(
r
t
1
1
1
0
1
0
(
(
(
(
(
(
9
(
(
0
3
n
2
n
e
9
9
0
0
e
U
i
2
3
r
2
2
3
3
x
a
a
t
p
e
c
H
o
D
B16 Cell Angiogenesis Inhibition
120
100
80
60
40
20
0
C.
30
150
100
50
31
26
29
Heparin
0
)
)
)
)
)
)
)
)
)
)
d
e
M
M
M
M
M
M
M
M
M
M
n
t
µ
µ
µ
9
µ
µ
µ
µ
0
µ
µ
l
0.1
1
10
a
e
3
7
3
3
3
7
.
1
3
(
3
3
3
3
.
.
.
(
r
t
1
1
1
0
1
0
(
(
(
(
(
(
9
(
(
0
3
n
2
n
e
9
9
0
0
e
U
i
Concentration (µM)
2
3
r
2
2
3
3
x
a
a
t
p
e
c
H
o
D
Figure 2. Screen of bioactivities of selected compounds. Panel A: Inhibition of metastasis and tumor growth in an
immunodeficient mouse xenograft model with human myeloma cells. See Methods (Figures S1 and S2 in the Supporting
Information) section for experimental details. On the left are untreated mice with malignant myeloma tumors containing
a luciferase reporter, 4 weeks after injection of tumor cells. A control mouse that was not injected with cells is included
(No. 5 from the left). On the right are mice injected with myeloma cells, and treated for 4 weeks with our heparanase
inhibitor 30 (600 g per day). Panel B: Inhibition of angiogenesis. The murine melanoma cell line B16 is capable of vascular
mimicry, a process similar to angiogenesis. The effect of the dendrimer HS glycomimetics 29 and 30 were tested to see if
they could prevent vascular mimicry in B16 cultures grown in a basement membrane extract (Cultrex BME) (see Methods).
Images of epithelial tubes or cords are shown in the upper part of Panel B, and a summary of the results after treatment for
18 h with 29 or 30 is shown on the lower part, presented as total number of tubes/cords and total length of tubes/cords (n=3
experiments; mean±SEM). Heparin was used as a negative control, and docetaxel (DTX) was used as a positive control for
inhibition of angiogenesis. Panel C: Inhibition of Factor Xa anticoagulant activity by HS glycomimetics (n=6 replicates;
mean±SD).
B.
A.
Figure 3. Screen of FGF signalling regulation by selected compounds. Panel A: No HS glycomimetic activation of
proliferation of BaF3 cells transfected with FGFR1c by FGF-1 or FGF-2. Left graph, proliferation activity of 1 nM FGF-1 in the
presence of increasing concentrations of heparin or compounds; Right, same as left graph but with 1 nM FGF-2. See Methods
section for experimental details. Panel B: Inhibition of heparin-induced activation of FGFR1c. FGF2 action is inhibited but
ACS Paragon Plus Environment

ACS Chemical Biology
Page 6 of 8
not FGF1 by selected compounds. Left, 100 μg/ml of compounds were added to BaF3 cells transfected with FGFR1c in the
presence of 1 nM FGF-1 and 3 μg/ml heparin. Cells exposed to no growth factor, 1 ng/ml IL-3 or FGF-1 alone are also shown.
Right, same as left graph but with 1 nM FGF-2. See Methods section for experimental details.
1
2
3
4
5
6
7
8
9
In summary, we have for the first time prepared chemically defined, single-entity polyvalent heparanase inhibitors
displaying simple sulfated sugars on dendritic cores. We have shown that these clusters inhibit heparanase with potency
equivalent to the comparator PG545, as well as inhibiting angiogenesis and FGF2, and they also completely lack off-target
anticoagulant activity and cell toxicity. The compounds reported here are a novel class of anticancer candidates with
additional potential applications in diseases where targeting of heparanase inhibition is expected to be beneficial, including
inflammatory disorders31, diabetic nephropathy32, fibrosis33 and viral pathogenesis34-35
.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
METHODS
All methods are described in the Supporting Information.
ASOOCIATED CONTENT
Supporting Information is available free of charge via the Internet on the ACS Publications website at http://pubs.acs.org.
Materials, methods, abbreviations, supplementary figures, detailed description of the synthetic procedures and NMR spectra
(PDF).
AUTHOR INFORMATION
Corresponding Author
*E-mail: Olga.Zubkova@vuw.ac.nz (O. V. Zubkova)
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
The authors thank H. Wong and Y. Lu for an excellent NMR and Mass Spectrometry service, and V. Ferro for the gift of PG545,
prepared according to the literature23.
REFERENCES
(1) Xu, D.; Esko, J. D., Demystifying Heparan Sulfate–Protein Interactions. Annu. Rev. Biochem. 2014, 83, 129-157.
(2) Bishop, J. R.; Schuksz, M.; Esko, J. D., Heparan sulphate proteoglycans fine-tune mammalian physiology. Nature 2007, 446, 1030-1037.
(3) Poon, S.; Lu, X.; Smith, R. A. A.; Ho, P.; Bhakoo, K.; Nurcombe, V.; Cool, S. M., Improved recovery from limb ischaemia by delivery of an affinity-isolated heparan
sulphate. Angiogenesis 2018, 21, 777–791.
(4) Sanderson, R. D.; Elkin, M.; Rapraeger, A. C.; Ilan, N.; Vlodavsky, I., Heparanase regulation of cancer, autophagy and inflammation: New mechanisms and targets
for therapy. FEBS J. 2017, 284, 42-55.
(5) Rivara, S.; Milazzo, F. M.; Giannini, G., Heparanase: a rainbow pharmacological target associated to multiple pathologies including rare diseases. Future Med.
Chem. 2016, 8, 647-680.
(6) Peterson, S. B.; Liu, J., Multi-faceted substrate specificity of heparanase. Matrix Biol. 2013, 32, 223-227.
(7) Arvatz, G.; Weissmann, M.; Ilan, N.; Vlodavsky, I., Heparanase and cancer progression: New directions, new promises. Hum. Vacc. Immunother. 2016, 12, 2253-
2256.
(8) Jia, L.; Ma, S., Recent advances in the discovery of heparanase inhibitors as anti-cancer agents. Eur. J. Med. Chem. 2016, 121, 209-220.
(9) Weissmann, M.; Arvatz, G.; Horowitz, N.; Feld, S.; Naroditsky, I.; Zhang, Y.; Ng, M.; Hammond, E.; Nevo, E.; Vlodavsky, I.; Ilan, N., Heparanase-neutralizing
antibodies attenuate lymphoma tumor growth and metastasis. Proc. Natl. Acad. Sci. USA 2016, 113, 704-709.
(10) Tyler, P. C.; Guimond, S. E.; Turnbull, J. E.; Zubkova, O. V., Single-Entity Heparan Sulfate Glycomimetic Clusters for Therapeutic Applications. Angew. Chem.
Int. Ed. 2015, 54, 2718-2723.
(11) Lundquist, J. J.; Toone, E. J., The Cluster Glycoside Effect. Chem. Rev. 2002, 102, 555-578.
(12) Scholefield, Z.; Yates, E. A.; Wayne, G.; Amour, A.; McDowell, W.; Turnbull, J. E., Heparan sulfate regulates amyloid precursor protein processing by BACE1, the
Alzheimer's β-secretase. J. Cell Biol. 2003, 163, 97-107.
(13) Patey, S. J.; Edwards, E. A.; Yates, E. A.; Turnbull, J. E., Heparin Derivatives as Inhibitors of BACE-1, the Alzheimer's β-Secretase, with Reduced Activity against
Factor Xa and Other Proteases. J. Med. Chem. 2006, 49, 6129-6132.
(14) Schwörer, R.; Zubkova, O. V.; Turnbull, J. E.; Tyler, P. C., Synthesis of a Targeted Library of Heparan Sulfate Hexa- to Dodecasaccharides as Inhibitors of β-
Secretase: Potential Therapeutics for Alzheimer’s Disease. Chem. Eur. J. 2013, 19, 6817-6823.
(15) Khamsi, J.; Ashmus, R. A.; Schocker, N. S.; Michael, K., A high-yielding synthesis of allylglycosides from peracetylated glycosyl donors. Carbohydr. Res. 2012, 357,
147-150.
(16) Hammond, E.; Li, C. P.; Ferro, V., Development of a colorimetric assay for heparanase activity suitable for kinetic analysis and inhibitor screening. Anal.
Biochem. 2010, 396, 112-116.
(17) Li, J.; Pan, Q.; Rowan, P. D.; Trotter, T. N.; Peker, D.; Regal, K. M.; Javed, A.; Suva, L. J.; Yang, Y., Heparanase promotes myeloma progression by inducing
mesenchymal features and motility of myeloma cells. Oncotarget 2016, 7, 11299-11309.
(18) Ramani, V. C.; Zhan, F.; He, J.; Barbieri, P.; Noseda, A.; Tricot, G.; Sanderson, R. D., Targeting heparanase overcomes chemoresistance and diminishes relapse in
myeloma. Oncotarget 2016, 7, 1598-1607.
(19) Ritchie, J. P.; Ramani, V. C.; Ren, Y.; Naggi, A.; Torri, G.; Casu, B.; Penco, S.; Pisano, C.; Carminati, P.; Tortoreto, M.; Zunino, F.; Vlodavsky, I.; Sanderson, R. D.;
Yang, Y., SST0001, a chemically modified heparin, inhibits myeloma growth and angiogenesis via disruption of the heparanase/syndecan-1 axis. Clin. Cancer Res. 2011,
17, 1382-1393.
(20) Racordon, D.; Valdivia, A.; Mingo, G.; Erices, R.; Aravena, R.; Santoro, F.; Bravo, M. L.; Ramirez, C.; Gonzalez, P.; Sandoval, A.; González, A.; Retamal, C.; Kogan,
M. J.; Kato, S.; Cuello, M. A.; Osorio, G.; Nualart, F.; Alvares, P.; Gago-Arias, A.; Fabri, D.; Espinoza, I.; Sanchez, B.; Corvalán, A. H.; Pinto, M. P.; Owen, G. I.,
Structural and functional identification of vasculogenic mimicry in vitro. Sci. Rep. 2017, 7, 6985.
ACS Paragon Plus Environment

Page 7 of 8
ACS Chemical Biology
(21) Berry, D.; Shriver, Z.; Venkataraman, G.; Sasisekharan, R., Quantitative assessment of FGF regulation by cell surface heparan sulfates. Biochem. Biophys. Res.
Commun. 2004, 314, 994-1000.
(22) Mathey-Prevot, B.; Nabel, G.; Palacios, R.; Baltimore, D., Abelson virus abrogation of interleukin-3 dependence in a lymphoid cell line. Mol. Cell. Biol. 1986, 6,
4133-4135.
(23) Ornitz, D. M.; Yayon, A.; Flanagan, J. G.; Svahn, C. M.; Levi, E.; Leder, P., Heparin is required for cell-free binding of basic fibroblast growth factor to a soluble
receptor and for mitogenesis in whole cells. Mol. Cell. Biol. 1992, 12, 240-247.
(24) Ferro, V.; Liu, L.; Johnstone, K. D.; Wimmer, N.; Karoli, T.; Handley, P.; Rowley, J.; Dredge, K.; Li, C. P.; Hammond, E.; Davis, K.; Sarimaa, L.; Harenberg, J.;
Bytheway, I., Discovery of PG545: A Highly Potent and Simultaneous Inhibitor of Angiogenesis, Tumor Growth, and Metastasis. J. Med. Chem. 2012, 55, 3804-3813.
(25) Dredge, K.; Hammond, E.; Davis, K.; Li, C. P.; Liu, L.; Johnstone, K.; Handley, P.; Wimmer, N.; Gonda, T. J.; Gautam, A.; Ferro, V.; Bytheway, I., The PG500 series:
novel heparan sulfate mimetics as potent angiogenesis and heparanase inhibitors for cancer therapy. Invest. New Drugs 2010, 28, 276-283.
(26) Dredge, K.; Brennan, T.; Brown, M. P.; Lickliter, J. D.; Bampton, D.; Hammond, E.; Lin, L.; Yang, Y.; Millward, M., An open-label, multi-center phase I study of
the safety and tolerability of the novel immunomodulatory agent PG545 in subjects with advanced solid tumors. J. Clin. Oncol. 2017, 35, 3083-3083.
(27) O'Reilly, E. M.; Roach, J.; Miller, P.; Yu, K. H.; Tjan, C.; Rosano, M.; Krause, S.; Avery, W.; Wolf, J.; Flaherty, K.; Nix, D.; Ryan, D. P., Safety, Pharmacokinetics,
Pharmacodynamics, and Antitumor Activity of Necuparanib Combined with Nab-Paclitaxel and Gemcitabine in Patients with Metastatic Pancreatic Cancer: Phase I
Results. Oncologist 2017, 22, 1429-e139.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(28) Galli, M.; Chatterjee, M.; Grasso, M.; Specchia, G.; Magen, H.; Einsele, H.; Celeghini, I.; Barbieri, P.; Paoletti, D.; Pace, S.; Sanderson, R. D.; Rambaldi, A.; Nagler,
A., Phase I study of the heparanase inhibitor Roneparstat: an innovative approach for multiple myeloma therapy. Haematol. J. 2018, 103, e469–e472.
(29) Zhou, H.; Roy, S.; Cochran, E.; Zouaoui, R.; Chu, C. L.; Duffner, J.; Zhao, G.; Smith, S.; Galcheva-Gargova, Z.; Karlgren, J.; Dussault, N.; Kwan, R. Y. Q.; Moy, E.;
Barnes, M.; Long, A.; Honan, C.; Qi, Y. W.; Shriver, Z.; Ganguly, T.; Schultes, B.; Venkataraman, G.; Kishimoto, T. K., M402, a Novel Heparan Sulfate Mimetic, Targets
Multiple Pathways Implicated in Tumor Progression and Metastasis. PLoS ONE 2011, 6, e21106.
(30) Hammond, E.; Handley, P.; Dredge, K.; Bytheway, I., Mechanisms of heparanase inhibition by the heparan sulfate mimetic PG545 and three structural
analogues. FEBS Open Bio. 2013, 3, 346-351.
(31) Goldberg, R.; Meirovitz, A.; Hirshoren, N.; Bulvik, R.; Binder, A.; Rubinstein, A. M.; Elkin, M., Versatile role of heparanase in inflammation. Matrix biol. 2013, 32,
234-240.
(32) Gil, N.; Goldberg, R.; Neuman, T.; Garsen, M.; Zcharia, E.; Rubinstein, A. M.; van Kuppevelt, T.; Meirovitz, A.; Pisano, C.; Li, J.-P.; van der Vlag, J.; Vlodavsky, I.;
Elkin, M., Heparanase Is Essential for the Development of Diabetic Nephropathy in Mice. Diabetes 2012, 61, 208-216.
(33) Secchi, M. F.; Crescenzi, M.; Masola, V.; Russo, F. P.; Floreani, A.; Onisto, M., Heparanase and macrophage interplay in the onset of liver fibrosis. Sci. Rep. 2017,
7, 14956.
(34) Thakkar, N.; Yadavalli, T.; Jaishankar, D.; Shukla, D., Emerging Roles of Heparanase in Viral Pathogenesis. Pathogens 2017, 6, 43.
(35) Supramaniam, A.; Liu, X.; Ferro, V.; Herrero, L. J., Prophylactic Antiheparanase Activity by PG545 Is Antiviral In Vitro and Protects against Ross River Virus
Disease in Mice. Antimicrob. Agents Chemother. 2018, 62, e01959-17.
Insert Table of Contents artwork here
ACS Paragon Plus Environment

ACS Chemical Biology
Page 8 of 8
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table of Contents
338x190mm (96 x 96 DPI)
ACS Paragon Plus Environment