1. Carbohydrate-based inducers of
cellular stress for targeting cancer cells
Fidelis Ndombera

2. WAYNE STATE UNIVERSITY CHEMISTRY DEPARTMENT
DETROIT, USA

3. Cancer metabolism generates high reactive oxygen species
O2
.-, H2O2, HO.
Nature, (2012) 491, 364

4. Redox balance control in cancer cells
Nature Reviews Drug Discovery (2013) 12, 931–947

5. ROS modulation as an anticancer strategy
normal cells cancer cells
ROS
level
Therapeutic
window
Cell death

6. Non-carbohydrate based ROS-modulating small molecules
Buthionine sulfoximine Gamma-glutamylcysteine synthetase inhibitor
Phenethyl isothiocyanate Activates NRF2/Phase II antioxidant enzymes
AAPS J. (2010) ; 12 (1): 87–97
Piperlongumine Depletes GSH by binding to Glutathione S-transferase pi
Nature. (2011) 475(7355): 231–234

7. Why design
Carbohydrate based ROS-modulating anti-cancer agents?
Position Emission Tomography
Scan image of lung cancer
2-deoxy-D-glucose 2-18Fluoro-2-deoxy-D-glucose
Oncogene (2006) 25, 4633–4646
• 250 mg/kg with radiotherapy
• 63 mg/kg with docetaxel
Strahlentherapie und Onkologie (2005), 181, 8,507-514
Cancer Letters (2014) 355, 176
hexokinase
Cancer Chemotherapy and Pharmacology (2013) 71, 2, 523-530

8. D-glucose-conjugates are tolerated by GLUT-1
Chem. Sci.(2013) 4, 2319
Paclitaxel

9. Small-molecule library of potential ROS inducers
46 small molecules
Bioorganic & Medicinal Chemistry Letters, Volume 26, Issue 5, 2016, 1452–1456

10. ROS Quantification using Dichloroflourescein Assay (DCF)
+ H2O2
Esterases
0.5mM 0.025mM 10mM
DCF Assay with H1299 Lung Cancer cell line
Excitation wavelength 485nm
Emission wavelength 535nm
0
5000
10000
15000
20000
25000
30000
35000
0 200 400 600
RFU
H2O2 concentration (µM)
DCF Assay: Hydrogen peroxide
5mins
30mins
60mins
4hrs

11. 0
50
100
150
200
250
A1a
A1b
A3
A4
A5a
A5b
A6
A7
A8
B1
B2
B3a
B3b
B7a
B7b
B8
C1
C2
C4a
C4b
C5
C7
D1
D2
E1
E2
E5
E6
F1
F6
G5
G8
H1
H3
H5
H7a
H7b
H8
I4a
I4b
I7
I8
J5
J7
K8
L8
None
200 uM Compound (Measurements in duplicates)
G8
B8
A8
Fluorescence(Fi/Fo)Carbohydrate-based small-molecule ROS screening in H1299
cells
H8
K8
L8

12. 0.5
1
1.5
2
2.5
0 25 50 75 100
Compound (μM)
O
OH
OHHO
NH
HO
A8
Fluorescence(Fi/Fo)
0.5
1
1.5
2
2.5
0 25 50 75 100
Compound (μM)Compound (μM)Compound (μM)Compound (μM)
Fluorescence(Fi/Fo)
ONH
HOOH
OH
C8
Concentration-dependent small-molecule ROS screening in
H1299 cells
0.5
1
1.5
2
2.5
0 25 50 75 100
Compound (μM)
Fluorescence(Fi/Fo)
O
OH
OH
NH
HO
HO
G8
0.5
1
1.5
2
2.5
0 25 50 75 100
Compound (μM)Compound (μM)Compound (μM)Compound (μM)
Fluorescence(Fi/Fo)
ONH
HO
OH
OHL8
Bioorganic & Medicinal Chemistry Letters, Volume 26, Issue 5, 2016, 1452–1456

13. 0.5
1
1.5
2
2.5
0 25 50 75 100
Compound (μM)Compound (μM)Compound (μM)Compound (μM)
0.5
1
1.5
2
2.5
0 25 50 75 100
Compound (μM)Compound (μM)Compound (μM)Compound (μM)
Fluorescence(Fi/Fo)
Fluorescence(Fi/Fo)
O NH
HO OH
OH
ONH
HOOH
OH
H8 K8
O OH
HO OH
OH
O OH
HO OH
OH
0.5
1
1.5
2
2.5
0 25 50 75 100
Compound (μM)
Fluorescence(Fi/Fo)
H K
0.5
1
1.5
2
2.5
0 25 50 75 100
Compound (μM)
Fluorescence(Fi/Fo)
NH2
8
Concentration-dependent small-molecule ROS
screening in H1299 cells

14. Carbohydrate-based small-molecule cytotoxicity screening in
H1299 cells
0
20
40
60
80
100
120
A1a
A1b
A3
A4
A5a
A5b
A6
A7
A8
B1
B2
B3a
B3b
B7a
B7b
B8
C1
C2
C4a
C4b
C5
C7
D1
D2
E1
E2
E5
E6
F1
F6
G5
G8
H1
H3
H5
H7a
H7b
H8
I4a
I4b
I7
I8
J5
J7
K8
L8
None
Viability(Vi/Vo,%)
100uM Compound (Measurement in duplicates)
ONH
HOOH
OH
O NH
HO OH
OH
H8 K8

15. Concentration-dependent small-molecule cytotoxicity
screening in H1299 cells
0
20
40
60
80
100
120
0 20 40 60 80 100
H8
K8
Compound (μM)
Viability(Vi/Vo,%)
-20
0
20
40
60
80
100
120
0 100 200 300 400 500
H2O2
DG
Compound (μM)
Viability(Vi/Vo,%)
H2O2
A B
IC50=75µM
IC50=80µM
IC50=60µM
ONH
HOOH
OH
O NH
HO OH
OH
H8 K8

16. K8A raises ROS and is cytotoxic to H1299 cells
0
20
40
60
80
100
120
0 100 200 300 400 500
K8A
Compound (μM)Compound (μM)
Viability(Vi/Vo,%)
Ac2O/Pyridine
IC50=25µM
Bioorganic & Medicinal Chemistry Letters, Volume 26, Issue 5, 2016, 1452–1456
0
20
40
60
80
100
120
0 50 100 150 200 250
K
KA
Compound (μM)
Viability(Vi/Vo,%)

17. Breast Cancer
CNS Cancer
Colon Cancer
Leukemia
Melanoma
Non-Small Cell Lung
Cancer
Renal Cancer
0
20
40
60
80
100
%GrowthInhibition
National Cancer Institute Cell line
% Growth Inhibition at 10uM K8A
K8A is cytotoxic in some NCI-60 cell lines
ONE DOSE DATA FROM NATIONAL CANCER INSTITUTE

18. Bioorganic & Medicinal Chemistry Letters, Volume 26, Issue 5, 2016, 1452–1456
K8A activates AMPK and stabilizes p53
HEK 293 cells
AMPK
P53
cell growth control

19. K8A induces ER stress at lower concentrations than 2DG
Bioorganic & Medicinal Chemistry Letters, Volume 26, Issue 5, 2016, 1452–1456
HEK 293 cells
• ER covers 12% of the cell’s volume.
• Site of protein synthesis & folding.
• Folds 1/3 of all proteins.
Glucose-regulated protein 78
is a biomarker of ER stress

20. Does K8A interfere with protein glycosylation?
N-acetyl glucosamine β-O-linked N-acetyl glucosamine protein
N-acetyl glucosamine with alkyne handle Clickable glycoprotein
Hexosamine Biosynthetic
pathway
HB Salvage pathway

21. K8A blocks protein glycosylation
K8A(uM) - - 30 30 50 100
GluNAckyne(20uM) - + - + + +
K8A

22. Does K8A inhibit an enzyme in hexosamine
biosynthetic salvage pathway?
PNAS 100,9116-9122(2003)
PNAS 104,2614-2619(2007)
PNAS 104, 16793-16797(2007)
Science 337,975-980
Nature Communications(2015)6:8468
DOI:10.1038/ncomms9468

23. Summary and conclusion
• Development of a library of simple N-aryl glycoside derivatives from unprotected sugars.
• Cellular ROS and cytotoxicity assays of this N-aryl-glycoside library led to identifying the
potential candidate (K8A) that activates AMPK, and stabilizes p53 in HEK293 cells, and
induce cytotoxicity with a higher potency than 2-DG in H1299 cells and other cell lines.
• K8A blocks protein glycosylation which is upregulated in Cancer.
• Target identification will be further investigated in future studies

24. ACKNOWLEDGEMENT
Young-Hoon Ahn lab