1. 
Biochemical Characterization of a Small Molecule Glycogen Synthase Activator
Yang Wen1, Yingsi Chen1, Qing Xiang1, Xiaolei Zhang1, Karen Rowan1, Kuo-Sen Huang1, David Mark1, Joseph A. Sergi3, David R. Bolin2, Andrée R. Olivier3
Discovery Technologies1, Discovery Chemistry2, Metabolic & Vascular Diseases3, Roche, 340 Kingsland St., Nutley, NJ, 07110
ENZYME ASSAY PROCEDURE: The enzymatic activity of GS
was measured at RT in polystyrene 384-well plates (BD Biosciences)
using a non-radioactive coupled assay in a total volume of 32 µl per
well. First an 8µl aliquot of Activator Solution (diluted compounds in
30 mM glycylglycine, pH 7.3, 40 mM KC1, 20 mM MgC12 , 9.2%
DMSO, with or without 20mM glucose-6-phosphate) was added to an
12µl aliquot of Substrate Solution (glycogen (4.32 mg/ml), 2.67 mM
UDP-glucose, 21.6 mM phospho(enol)pyruvate and 2.7 mM NADH
in 30 mM glycylglycine, pH 7.3 buffer) in the appropriate wells. The
reaction was then started by adding 12µl of Enzyme Solution
(glycogen synthase (16.88µg/ml), pyruvate kinase (0.27 mg/ml),
lactate dehydrogenase (0.27 mg/ml) in 50 mM Tris-IICl, pH 8.0, 27
mM DTT and bovine serum albumin (BSA, 0.2 mg/ml)) to the
appropriate wells, mixed and incubated at room temperature. The
conversion rate of NADH to NAD was measured every 3 min over a
period of 15 minutes at Abs 340 nm on an Envision reader (Perkin
Elmer). The enzyme activity (with or without compound) was
calculated by the reaction rate per minute. EC50 is defined as the
compound concentration that is needed to give 50% maximum
activation.
INTRODUCTION
The key step in the synthesis of glycogen is the addition of UDP-
glucose to the growing glycogen chain, and is catalyzed by the
enzyme glycogen synthase (GS). There is substantial clinical and
genetic evidence linking GS to various metabolic disorders. Basal and
insulin-stimulated GS activities as well as glycogen content in muscle
cells from diabetic subjects are significantly lower than in cells from
lean non-diabetic subjects. Decreased muscle glycogen synthesis can
promote atherogenic dyslipidemia in IR patients. Human mutations in
muscle GS, in the GYS1 gene promotor (Xba1), have been identified
that result in decreased GS activity/levels and are associated with
insulin resistance in some cohorts. These subjects also have increased
risk for cardiovascular disease. Thus, an activator of GS has potential
as a novel therapeutic agent for the treatment of metabolic diseases,
such as type 2 diabetes (T2D) and cardiovascular diseases.
GLYCOGEN SYNTHASE ASSAY
CONCLUSIONS
GSA3 activates glycogen synthase specifically
(Glycogen)n + UDPG
Glycogen Synthase
(Glycogen)n+1 + UDP
UDP + Phospho(enol)pyruvate Pyruvate + UTP
Pyruvate Kinase
Pyruvate + NADH
Lactate Dehydrogenase
Lactate + NAD
ENZYME ASSAY PRINCIPLE
Although GS sample has GP activity and PP 1 activity, these
activities are not involved in GSA3 activation
Decreased glycogen synthesis is a common defect in T2D and obese patients. In an attempt to ameliorate this defect we searched for compounds that can
directly activate the rate limiting enzyme GS. Small molecule Glycogen Synthase Activators (GSA) were identified in a high throughput screen using a non –
radiometric coupled assay. After high throughput screening and lead optimization efforts, GSA3 was generated with improved activation potency. The
biochemical characterization of GSA3 is presented.
µM GSA3
0.0001 0.01 1 100
GSActivitynmolesmin-1µg-1
5
10
15
20
25
-2 0 2
0
5
10
w/o UDPG
w/ UDPG
log GSA3 µM
RateµM/min
UDPG vs GSA3
Double-Reciprocal Plot
-0.075 0.075 0.150 0.225
250
500
750
       

  

 cpd - 0.39 uM
cpd - 0.195 uM
cpd - 0.024 uM
cpd - 0.012 uM
cpd - 0.006 uM
no cpd
cpd - 1.5625 uM
cpd - 0.098 uM
cpd - 0.78 uM
cpd - 0.049 uM
1/UDPG (M-1
)
1/Rate
(mM/min-1
)
-2 -1 0 1 2
-5
0
5
10
15
theoretical sum of GSA3 & G6P
GSA3 + G6P
GSA3 Alone
log GSA3 µM
RateµM/min
PP1 Activity in GS sample
0 5 10 15 20
0
200000
400000
600000
800000
GS Sample Alone
GS Sample w/ GSA3
[Protein] µg/ml
Fluorescence
GSA3 Dose Response in
GS assay w/ and w/o PP1 inhibitor
-2 0 2
0
5
10
GSA3 Alone
GSA3 + PP1 inhibitor
log GSA3 µM
RateµM/min
GP Activity in GS Sample
0 5 10 15 20
0.0
0.5
1.0
1.5
GS Sample Alone
GS Sample w/ GSA3
[Protein] µg/ml
Abs340nm
GSA3 Dose Response in
GS Assay w/ and w/o GP inhibitor
-2 0 2
0
5
10
GSA3 Alone
GSA3 + GP inhibitor
log GSA3 µM
RateµM/min
GSA3 increases the enzyme affinity for substrate UDPG ( Vmax Km) and does not compete with UDPG in GS binding
Synergistic effect of G6P and GSA3 on GS activation
-5 0 5 10 15 20 25
8
10
12
14
GSA3 (M)
ApparentVmax(M/min)
0 20 40 60 80 100
0
8
16
24
32
GSA3 (M)
ApparentKm(mM)
 Lead compound GSA3 activates the GS enzyme specifically.
 GSA3 and substrate UDPG bind to separate binding sites on the GS
protein.
 GSA3 and G6P act synergistically in activation of the GS enzyme. It
decreases the Km for the substrate UDPG without increasing the Vmax.
 Although PP1 and GP activities were detected in the GS sample,
GSA3 does not activate PP1 or GP. Inhibitors of PP1 or GP do not
affect the activation of GS by GSA3.
ACKNOWLEDGEMENTS
The authors would like to thank Chao-Min Liu, Charles Belunis,
David Weber and Ueli Gubler for cloning, expression and purification
of human recombinant GYS1 used in these studies; and Shirley Li for
providing protocols and materials of the PP1 and GP assays. We
would also like to thank Janet Diratsaoglu, Peggy Borgese and
Beverley Simko for preparing the compounds.
RESULTS
GSA3 shows nice dose
response in the coupled
GS assay. However when
the substrate UDPG was
absent, no GS activity
was detected regardless of
GSA3 concentration. The
result of the breakdown
assay further confirms
that GSA3 activates GS
specifically.
The GS sample used in our study was tested for Protein
Phosphatase 1 (PP1) and Glycogen Phosphorylase (GP) activities.
The starting concentration of the GS sample in these assays was 3
folds higher than what’s used in the GS coupled assay. Although
PP1 and GP activities were detected in the GS sample, the
presence of GSA3 does not enhance these activities. This result
indicates that the activation of GS by GSA3 was specific. PP1
inhibitor and GP inhibitor did not change the activator behavior
of GSA3 in the GS assay which further confirm that the GS
activity we observed didn’t come from other sources.
GSA3 acts synergistically with G6P to decrease the Km for UDPG and increase the catalytic efficiency of the enzyme without a further increase in Vmax.
GSA3 activates GS
specifically according
to the result of direct
radiometric assay
m e a s u r i n g t h e
incorporation of 14C-
UDPG into glycogen.
0 30 60 90 120 150
5
10
15 25 uM
12.5 uM
6.25 uM
3.125 uM
1.563 uM
0.781 uM
0.391 uM
0.195 uM
0.098 uM
0.049 uM
0.024 uM
0.006 uM
no compound
0.012 uM
UDPG (M)
Rate(M/min)
•GSA3 does not compete with UDP-Glucose (UDPG) in GS binding
•GSA3 increases the enzyme affinity for substrate UDPG
•GSA3 increases the Vmax for UDPG
G6P vs. GSA3 Double-Reciprocal Plot
-0.5 0.5 1.0 1.5 2.0
100
200
300
400
50 (uM)
25 (uM)
12.5 (uM)
6.25 (uM)
3.125 (uM)
1.563 (uM)
0.781 (uM)
0.391 (uM)
1/G6P (mM-1)
1/Rate(mM/min-1
)
0 10 20 30 40
0
5
10
15 50 (uM)
25 (uM)
12.5 (uM)
6.25 (uM)
3.125 (uM)
1.563 (uM)
0.781 (uM)
0.391 (uM)
0.195 (uM)
no compound
G6P (mM)
Rate(M/min)
0 10 20 30 40 50
0.0
0.5
1.0
1.5
2.0
2.5
GSA3 (M)
ApparentKm(mM)