Optimizing glucokinase activator binding kinetics to lower in vivo hypoglycemia risk

Article abstract of DOI:10.1039/c4md00027g

Activation of glucokinase represents a promising strategy for the treatment of type 2 diabetes; however, drug candidates have failed in clinical trials due to narrow therapeutic index between glucose-lowering efficacy and hypoglycemia. Described herein is a novel strategy for the design of next generation glucokinase activators with increased therapeutic index, which involves the optimization of activator-enzyme binding kinetics (k_on, k _off). This approach is based on the idea that activator binding kinetics are relevant to pharmacodynamics since the affinity of activator binding to glucokinase is cooperative with glucose such that, the rate at which an activator dissociates may influence the enzyme's sensitivity to changes in physiological glucose concentrations. This study provides a compelling example of using fast-off binding kinetics for developing safe and effective activator drugs targeting glucokinase.

Full text of DOI:10.1039/c4md00027g

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Optimizing glucokinase activator binding kinetics
to lower in vivo hypoglycemia risk†
Cite this: Med. Chem. Commun., 2014,
5, 802
Kris A. Borzilleri,a Jeffrey A. Pfefferkorn,b Angel Guzman-Perez,e Shenping Liu,a
Xiayang Qiu,a Boris A. Chrunyk,a Xi Song,a Meihua Tu,b Kevin J. Filipski,b Robert Aiello,c
David R. Derksen,a Francis J. Bourbonais,a James Landro,f Patricia Bourassa,c
Theresa D'Aquila,d Levenia Baker,g Nicole Barrucci,c John Litchfield,a Karen Atkinson,a
b
a
*
Timothy P. Rolph and Jane M. Withka
Activation of glucokinase represents a promising strategy for the treatment of type 2 diabetes; however,
drug candidates have failed in clinical trials due to narrow therapeutic index between glucose-lowering
efficacy and hypoglycemia. Described herein is a novel strategy for the design of next generation
glucokinase activators with increased therapeutic index, which involves the optimization of activator-
enzyme binding kinetics (kon, koff). This approach is based on the idea that activator binding kinetics are
relevant to pharmacodynamics since the affinity of activator binding to glucokinase is cooperative with
glucose such that, the rate at which an activator dissociates may influence the enzyme's sensitivity to
changes in physiological glucose concentrations. This study provides a compelling example of using
fast-off binding kinetics for developing safe and effective activator drugs targeting glucokinase.
Received 21st January 2014
Accepted 18th April 2014
DOI: 10.1039/c4md00027g
www.rsc.org/medchemcomm
insulin therapy, the incidence of hypoglycemia (glucose < 50 mg
Introduction
dLꢀ1) was 14–16% in the active treatment arms versus 8% for
the placebo group. Furthermore, four patients on MK-0941
discontinued from the study due to hypoglycemia and one
subject in the 20 mg group experienced an episode of severe
hypoglycemia requiring medical assistance.4a Additionally,
during this same study of MK-0941, tachyphylaxis was observed
aer 14 weeks of treatment.4a A similar loss of efficacy was also
recently reported in a phase 2 study of GKA AZD-1656.4b The
explanation for this tachyphylaxis is currently unclear, and
studies with additional activators in various patient populations
are needed to further investigate the issue.
Given the potential therapeutic limitations and compliance
issues (e.g. dose titration and/or dosing with meals) imposed by
the risk of GKA-induced hypoglycemia, there is continued
interest in drug design strategies to develop next generation
agents with reduced risk. Biochemically, glucokinase catalyzes
the conversion of glucose to glucose-6-phosphate, which is the
rst and rate limiting step in glycolysis and glycogen synthesis.
Glucokinase functions as a key regulator of glucose homeo-
stasis and small molecule allosteric activators of this enzyme
have been shown to reduce blood glucose levels by enhancing
glucose-stimulated insulin secretion in the pancreas while also
increasing glucose uptake and reducing glucose production in
the liver.1,2 The development of glucokinase activators (GKAs)
has been an active area of pharmaceutical research with several
candidates advancing to early clinical studies. However, while
these GKAs have shown promising early efficacy in T2DM
patients, there has also been signicant attrition driven by
narrow therapeutic windows against hypoglycemia as well as
concerns about durability and adverse effects on plasma and
hepatic lipids.3,4 For example, during a phase 2 study of GKA
MK-0941 (10–40 mg, TID) conducted in T2DM patients on basal
aPzer Worldwide Research and Development, Eastern Point Road, Groton, CT 06340,
USA. E-mail: jane.m.withka@pzer.com
bPzer Worldwide Research and Development, 620 Memorial Drive, Cambridge, MA
It has a physiologically optimal affinity for glucose (Km or S0.5
¼
8 mM) and has been shown to demonstrate positive coopera-
tivity of glucose binding,5 facilitated by multiple conformations
of this monomeric protein in solution.6,7 High resolution
structures of glucokinase complexed with several activator
02139, USA
cKaros Pharmaceuticals, Science Park, New Haven, CT 06511, USA
dPurdue University, West Lafayette, Indiana 47907, USA
eAmgen, 360 Binney Street, Cambridge, MA 02142, USA
f412 Maple Avenue, Cheshire, CT 06410, USA
˚
chemotypes revealed an allosteric binding site, 20 A remote
from the catalytic site. Activators of glucokinase have been well
characterized and are known to modulate enzyme activity by
two parameters a and b, which are dened by a decrease in Km
g151 Concord Street, Newton, MA 02462, USA
† Electronic supplementary information (ESI) available: SPR proles for
compounds 6 and 15, KD and EC50 correlation, comparison of human and rat
off rates for select activators, synthetic protocols for compounds 3, 5, 6 and 11.
See DOI: 10.1039/c4md00027g
and an increase in Vmax
,
respectively.8–10 Previously, we
802 | Med. Chem. Commun., 2014, 5, 802–807
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Table 1 Binding kinetics, potency and activation profiles of glucokinase activators 1–17
kon ꢂ 105 ꢁ SEa koff ꢂ 10ꢀ3 ꢁ SEa KD ꢁ SEa
EC50
(nM)
[GKA]chypoglycemia <5
[GKA]efficacy
b
Dissociation
Cmd Ref. Structure
t1/2 ꢁ SEa (s) (Mꢀ1 sꢀ1
)
(sꢀ1
)
(nM)
ab
bb
1
2
15
16
980 ꢁ 22
430 ꢁ 4
2.41 ꢁ 0.07
0.71 ꢁ 0.02
1.61 ꢁ 0.02
2.93 ꢁ 0.10
2.16 ꢁ 0.05
9.5 0.05 1.27 Yes
16.2 0.05 1.05 Yes
7.46 ꢁ 0.17
9.75 ꢁ 0.41
3d,g
294 ꢁ 5
2.36 ꢁ 0.04
5.61 ꢁ 0.04
6.05 ꢁ 0.02
6.57 ꢁ 0.04
7.01 ꢁ 0.05
2.42 ꢁ 0.11
10.8 ꢁ 0.1
86.4 ꢁ 1.1
17.5 ꢁ 0.2
89.9 ꢁ 1.4
9.4 0.08 0.92 Yes
4
17
123.5 ꢁ 0.8
114.6 ꢁ 0.5
105.5 ꢁ 0.7
98.9 ꢁ 0.7
5.17 ꢁ 0.04
0.70 ꢁ 0.01
3.76 ꢁ 0.03
0.78 ꢁ 0.01
28
121
24
0.1
0.86 Yes
5e,g
6d,g
7
0.06 1.2
Yes
0.07 0.85 Yes
0.04 1.28 Yes
9
26
8
9
18
20
94.8 ꢁ 0.3
81.9 ꢁ 0.8
2.85 ꢁ 0.03
0.60 ꢁ 0.01
7.31 ꢁ 0.02
8.46 ꢁ 0.08
25.6 ꢁ 0.3
37
0.05 1.22 Yes
141.5 ꢁ 2.8
191
0.09 0.86 Lack of efficacyf
10
19
69.8 ꢁ 0.6
21.1 ꢁ 0.2
15.8 ꢁ 0.3
4.65 ꢁ 0.11
3.40 ꢁ 0.07
2.05 ꢁ 0.05
9.93 ꢁ 0.08
32.9 ꢁ 0.3
43.9 ꢁ 0.8
21.4 ꢁ 0.5
96.8 ꢁ 2.2
218.4 ꢁ 6.6
52
120
364
0.08 0.82 Yes
11d,g
0.08
1
Lack of efficacyf
12
21
0.04 1.73 Yes
13
14
15
16
20
9
14.7 ꢁ 0.1
12.3 ꢁ 0.2
9.7 ꢁ 0.1
8.4 ꢁ 0.1
4.12 ꢁ 0.07
3.22 ꢁ 0.09
3.79 ꢁ 0.07
1.94 ꢁ 0.04
47.0 ꢁ 0.3
56.3 ꢁ 0.8
72.7 ꢁ 0.7
82.9 ꢁ 0.6
96.6 ꢁ 0.4
114.1 ꢁ 2.1
174.8 ꢁ 5.3
189.2 ꢁ 3.7
427.3 ꢁ 8.8
164.9 ꢁ 1.2
114
210
174
391
80
0.12 0.82 No
0.09 0.96 No
9
0.1
0.87 No
20
10
0.11 0.8
No
17
7.2 ꢁ 0.03 5.86 ꢁ 0.03
0.04 1.33 No
a
Dissociation t1/2 ¼ ln 2/koff; kinetic parameters (kon, koff) were obtained by a global t using three concentrations in duplicate; KD determined by
the standard equation, KD ¼ Koff/Kon; SE¼ standard error. b Potency (EC50) and activation prole (a, b) determined as described and reported as the
c
mean of n > 2 independent determinations.9 Preclinical in vivo efficacy and hypoglycemia safety evaluated in Sprague-Dawley rat as described.4
d
e
f
Single stereoisomer, absolute stereochemistry not determined. Racemic. Activator did not achieve >20% AUC reduction during OGTT at
highest dose (100 mg kgꢀ1) evaluated. g Synthetic protocols for compounds 3, 5, 6, and 11 are described in ESI.†
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published an experimental assessment of the hypoglycemic risk
associated with different a,b activation proles and successfully
identied a “partial activator” that demonstrated favorable
efficacy and minimal hypoglycemia risk in preclinical studies.9
As an extension of this work, we also began to consider how
activator-enzyme binding kinetics might inuence GKA resi-
dency time on the enzyme and inuence hypoglycemia safety.11
For many enzyme systems, slow off rates and long dissociative
half-lives (a direct measure of residence times) have been shown
to improve target selectivity and reduce off-target safety
risks.12,13 However in the case of GKAs there is positive cooper-
ativity between activator and glucose binding to the enzyme
making the apparent potency of an activator dependent on
glucose concentrations.14 As a consequence of this prole, the
rate at which an activator dissociates from the enzyme may play
a critical role in the ability of glucokinase to rapidly respond to
changing physiological glucose levels hence inuencing hypo-
glycemic risk. Finally, while durability of GKA efficacy is not the
focus of the current work, it should be acknowledged that
understanding differences in activator binding kinetics could
have additional, longer term, benet for understanding dura-
bility differences between activators.
Fig. 1 Kinetic plot showing relationship between off rates (koff), on
rates (kon) and binding affinities (KD) represented on the diagonal axis
for compounds in Table 1, and determined in SPR. Off rates and on
rates are plotted as logarithmic values. Only efficacious compounds
are represented on plot.
The inuence of binding kinetics on hypoglycemic risk was vitro binding kinetics to in vivo pharmacodynamic studies in
investigated using a series of structurally diverse GKAs span- rats.
ning a range of in vitro potencies (EC50 ¼ 10–391 nM) for which,
To obtain a pharmacokinetic/pharmacodynamic (PK/PD)
a detailed assessment of binding kinetic, enzyme activation and assessment of postprandial efficacy and hypoglycemia risk,
in vivo pharmacodynamic proles were determined. Speci- these activators were evaluated in Wistar rats during an oral
cally, the binding affinities and kinetics of these activators were glucose tolerance test (OGTT) as described previously.9 To
characterized against human recombinant glucokinase using evaluate efficacy, an oral glucose challenge (2 g kgꢀ1) was
surface plasmon resonance (SPR) as shown in Table 1. To administered 60 min post dose and the effect of the activator at
enable this experiment, biotinylated glucokinase was expressed reducing the glucose AUC excursion during a hyperglycemic
in E. coli, puried as previously described8,10 and captured onto state was determined. [GKA]efficacy was dened as concentration
a streptavidin sensor chip to levels ranging from 5000–6000 of unbound (free) activator affording a 20% reduction in
response units (RU). SPR binding experiments were carried out glucose AUC during an OGTT. [GKA]hypoglycemia was dened as
in duplicate for three activator concentrations with 3 fold the concentration of unbound (free) activator reducing fasting
dilution and in the presence of a saturating glucose concen- plasma glucose to <60 mg dLꢀ1. The hypoglycemic risk assess-
tration of 50 mM glucose (representative SPR sensorgrams ment was characterized by a therapeutic index (TI) <5 as
shown in ESI Fig. S1† for compounds 6 and 15). The activator calculated by [GKA]hypoglycemia/[GKA]efficacy and listed as “no” or
concentrations run in SPR were based on the EC50 with the top “yes” hypoglycemia in Table 1. A TI of 5 was selected to offer a
concentration chosen to be several fold over the EC50 to achieve reasonable margin of hypoglycemic safety relative to the Cmin to
saturation. Activator binding kinetics to glucokinase as a Cmax pharmacokinetic exposure range for a typical glucokinase
function of glucose concentrations were attempted but could activator. While the efficacy and hypoglycemia data from in vivo
not be rigorously determined in SPR due to conformational preclinical models may not directly translate to diabetic
instability of glucokinase at low glucose concentrations.
patients, we postulate that activators which are more efficacious
To validate our SPR results obtained at 50 mM glucose, we in a hyperglycemic state and cause less hypoglycemia in a
compared binding affinity (KD) data to activator potency (EC50), euglycemic state are likely to afford a wider therapeutic index in
which was dened as the ligand concentration required to show clinical studies.
a half maximal reduction in Km.9 Determination of binding
Kinetic studies for these activators binding to glucokinase,
affinities (KD) for compounds in Table 1 revealed a reasonable resulted in on rates (kon) which ranged from 0.70 ꢂ 105 to 9.75
correlation with compound potency (ESI Fig. S2†). Our data also ꢂ 105 (Mꢀ1 sꢀ1) while off rates (koff) ranged from 0.71 ꢂ 10ꢀ3 to
indicates that potency is predominantly driven by binding off 96.6 ꢂ 10ꢀ3 (sꢀ1) providing a range of dissociative half-lives
rates (Fig. 1), which can be readily converted to dissociative half- from 980 to 7.2 s. If we consider the binding kinetics of these
lives to assess its inuence on hypoglycemia. SPR binding compounds, shown as a kinetic plot in Fig. 1, off rates slower
kinetics were also determined against rat recombinant gluco- than approximately 4.4 ꢂ 10ꢀ2 sꢀ1 typically result in preclinical
kinase for compounds 6, 7, 15 and showed excellent correlation hypoglycemia with TIs < 5 despite similar binding affinities (KD)
with those determined for human glucokinase (Table S1†), in some cases. In our previous work, structural activity rela-
thereby providing additional condence in the translation of in tionships for a series of 2-substituted benzofurans indicated in
804 | Med. Chem. Commun., 2014, 5, 802–807
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vitro efficacy and a reduced risk of hypoglycemia for partial favorable a, b enzymatic proles yet these compounds also
activators with a moderate effect on Km, indicated by a values in exhibited unfavorable pharmacodynamic proles reected by
the range of 0.05–0.2 and a lesser effect on Vmax with b values in preclinical hypoglycemia TI < 5 and shown as red, lled circles
the range of 0.8–1.2.9 In the current study, compounds 1, 7, 8, in Fig. 2. This analysis clearly indicates the need for an addi-
and 12 in Table 1 and shown as red squares in Fig. 2, displayed tional preclinical predictive parameter, beyond a and b values,
unfavorable a, b proles resulting in low therapeutic indexes to mitigate hypoglycemia risk. For compounds 13–17 with
(TI) against hypoglycemia as predicted based upon previous reduced hypoglycemia risk (TI > 5), shown in green, the majority
work.9 Importantly, compounds 2–6 and 10 were identied with of them have favorable a, b proles in addition to relatively
short dissociation times. Based on this analysis, an upper limit
to the off rate of approximately 5 ꢂ 10ꢀ2 sꢀ1 or faster in
conjunction with favorable a, b proles are proposed to mini-
mize hypoglycemia risk.
The predictive value of including in vitro target residence
time as key criteria in advancing GKAs is illustrated by the in
vivo proles of two activators, compounds 6 and 15 in Fig. 3.
Both compounds show dose dependent glucose lowering with
oral dosing from 3–100 mg kgꢀ1. However, in the case of
compound 6, the free concentration of the activator normalized
to its EC50, which results in an efficacious reduction in plasma
glucose, is very close to the concentration for which hypogly-
cemia is induced. Although the dissociation half-life of
compound 6, equal to 105.5 s, is considered short compared
with those of many known drugs,12 it is still not short enough to
allow for rapid dissociation of the compound from the receptor
in response to highly varying glucose levels. This effect is
Fig. 2 3D plot showing correlation of alpha a (Km), beta b (Vmax) and
particularly critical for glucokinase which displays cooperative
binding of activators with increasing glucose levels, resulting in
dissociative half-life (t s), plotted on a logarithmic scale for effica-
½
cious compounds in Table 1. Compounds shown in red presented with
greater affinities, decreased off rates and longer residence
preclinical hypoglycemia with TIs <5 while those in green had TIs > 5.
times. As a result, the therapeutic windows are extremely
narrow. In the case of compound 15, the efficacious concen-
tration, indicated by the green line, is signicantly less than
Compounds with favorable a, b profiles are shown as circles and those
with unfavorable profiles are shown as squares (see ref. 9 for definition
and further discussion of favorable and unfavorable a, b).
Fig. 3 Potency (EC50), a/b profiles, dissociative half-lives, therapeutic index (left), dose dependent effect on plasma glucose following a single
dose at 3, 10, 30 and 100 mg kgꢀ1 during an oral glucose tolerance test (OGTT) (middle). Glucose excursion data expressed as means ꢁ standard
error (n ¼ 7). PK/PD plots (right) describing plasma glucose AUC reduction following OGTT in green and fasting plasma glucose reduction in red
vs. compound concentration normalized by the EC50 for compounds 6 and 15. Data for compound 15 is reprinted with permission from the
Royal Society of Chemistry.
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that predicted to result in hypoglycemia as indicated by the red
line. Although the a, b enzymatic proles are similar for both
compounds, the residence time for compound 15 is an order of
magnitude less than that for compound 6. Based on a favorable
preclinical efficacy and safety prole, compound 15 was
advanced into clinical trials and is currently in phase 2 studies
for the treatment of T2DM.
improves fasting and postprandial glucose in patients with
type 2 diabetes mellitus (T2DM). 71st American Diabetes
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This study suggests that optimizing the window between
efficacy and hypoglycemia safety for GKAs will require careful
attenuation of both enzyme activation proles (Km, Vmax effects)
as previously reported9 as well as activator binding kinetics and
more specically, off rates and resultant residence times on the
enzyme. Due to the positive cooperativity between activator
potency (EC50) and glucose concentrations as previously repor-
ted9 and the correlation of EC50 to KD/koff in this work, we
believe that at saturating glucose concentrations, activators will
display their slowest inherent off rates. When GKAs start to
lower in vivo glucose levels, they will have even faster target
dissociation rates, thus reducing their glucose-lowering efficacy
and limiting the risk of a hypoglycemic event. Consideration of
off rate parameters and residence times may be particularly
important for GKAs that have also been recently shown to
activate glucokinase in a glucose independent manner.22 The
strategy described here provides a successful example of using
fast-off binding kinetics to optimize therapeutic index for
glucokinase activators, which may have broader implications
for other enzyme activator or inhibitor approaches.
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