Discovery of MK-8722: A Systemic, Direct Pan-Activator of AMP-Activated Protein Kinase

Article abstract of DOI:10.1021/acsmedchemlett.7b00417

5′-Adenosine monophosphate-activated protein kinase (AMPK) is a key regulator of mammalian energy homeostasis and has been implicated in mediating many of the beneficial effects of exercise and weight loss including lipid and glucose trafficking. As such,

Full text of DOI:10.1021/acsmedchemlett.7b00417

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Letter
Discovery of MK-8722: A Systemic, Direct Pan-
Activator of AMP-Activated Protein Kinase
Danqing Feng, Tesfaye Biftu, F. Anthony Romero, Ahmet Kekec, James Dropinski, Andrew Kassick,
Shiyao Xu, Marc M Kurtz, Anantha Gollapudi, Qing Shao, Xiaodong Yang, Ku Lu, Gaochao Zhou, Daniel
Kemp, Robert W Myers, Hong-Ping Guan, Maria E. Trujillo, Cai Li, Ann Weber, and Iyassu K Sebhat
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.7b00417 • Publication Date (Web): 01 Dec 2017
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Discovery of MK-8722: A Systemic, Direct Pan-Activator of
AMP-Activated Protein Kinase.
Danqing Feng1, Tesfaye Biftu1, F. Anthony Romero1, Ahmet Kekec1, James Dropinski1, Andrew Kas-
sick1, Shiyao Xu2, Marc M. Kurtz3, Anantha Gollapudi4, Qing Shao4, Xiaodong Yang5, Ku Lu5, Gao-
chao Zhou5, Daniel Kemp5, Robert W. Myers5, Hong-Ping Guan5, Maria E. Trujillo4, Cai Li5, Ann We-
ber1 and Iyassu K. Sebhat1*.
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1Medicinal Chemistry, 2PPDM Preclinical ADME, 3In Vitro Pharmacology, 4In Vivo Pharmacology and 5Biology-Discovery
Departments, Merck Research Laboratories, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA.
KEYWORDS: AMPK, activator, T2DM, ACC, liver-selectivity.
ABSTRACT: AMP-Activated protein kinase (AMPK) is a key regulator of mammalian energy homeostasis and has been implicat-
ed in mediating many of the beneficial effects of exercise and weight loss including lipid and glucose trafficking. As such the en-
zyme has long been of interest as a target for the treatment of Type 2 Diabetes Mellitus (T2DM). We describe the optimization of
β1-selective, liver-targeted AMPK activators and their evolution into systemic pan-activators capable of acutely lowering glucose
in mouse models. Identifying surrogates for the key acid moiety in early generation compounds proved essential in improving β2-
activation and in balancing improvements in plasma unbound fraction while avoiding liver sequestration.
Type 2 Diabetes Mellitus (T2DM) is a chronic, progressive
disease characterized by insulin resistance leading to reduced
peripheral glucose uptake and increased hepatic glucose pro-
duction. The incidence of T2DM has reached epidemic pro-
portions with 642 million people projected to suffer from the
disease by 2040.1
tions appear to result in intrinsic activation of AMPK.7,8 It is
unclear which clinical manifestations of PRKAG2 cardiomyo-
pathy have developmental origins, but cardiac safety is a ma-
jor concern for the therapeutic application of systemic AMPK
activators.
Several classes of antihyperglycemic agents are prescribed
for the treatment of T2DM either singly or in combination
(e.g. sulfonylureas, metformin, PPARγ-selective agonists,
GLP-1 analogs, DPP-4 inhibitors, SGLT2 inhibitors, insulin
and insulin analogs). Despite the available options, most dia-
betic patients fail to achieve their therapeutic goal. In addi-
tion, many current therapies have significant limitations and
liabilities such as hypoglycemia, weight gain, edema, frac-
tures, lactic acidosis and gastrointestinal intolerance.2
Exercise, combined with weight loss, is a powerful means to
lower blood glucose levels in T2DM patients and forms the
foundation for effective treatment and disease management.3
Numerous beneficial effects of exercise are thought to be me-
diated through 5'-adenosine monophosphate-activated protein
kinase (AMPK).4 These include alterations in cellular fuel
utilization as well as changes in gene transcription.
Figure 1. Structure of A-769662
Despite a growing understanding of the link between
AMPK and exercise and the myriad potential applications of
an AMPK activator, 7 to date, there have been few reports of
small molecule direct activators.9,10 This likely reflects the
complexity of the enzyme as well as, in our case (and presum-
ably others), the failure of traditional high throughput screen-
ing campaigns to identify activator hits. Many of the existing
small molecule tools are indirect activators that work by in-
creasing the AMP/ATP ratio in cells. By far the most widely
used direct activator is the prodrug AICAR (1a), which is 5’-
phosphorylated intracellularly to the AMP-mimetic ZMP.
Unfortunately, the compound has poor selectivity and is rapid-
ly cleared in vivo. A-769662 (1b) was developed by Abbott
and has stood as the most widely investigated drug-like of the
AMPK is a Ser/Thr protein kinase that serves as the primary
mechanism for maintenance of energy homeostasis in eukary-
otic cells.5 The enzyme is a heterotrimer comprised of a cata-
lytic α-subunit (2 isoforms), a β-subunit (2 isoforms) and a
regulatory γ-subunit (3 isoforms); it is activated by changes in
cellular adenine nucleotide or calcium levels.6
However, there are potential safety concerns associated with
AMPK activation. Mutations in the AMPK γ2-subunit are
found in a percentage of Wolff-Parkinson-White syndrome
patients, leading to PRKAG2 cardiomyopathy. These muta-
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We developed robust in vitro assays and in vivo pharmaco-
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small molecule, non-nucleoside activators. A-769662 selec-
tively activates the β1-containing AMPK complexes and does
not activate the 6 β2-containing complexes.11 The molecule
also suffers from some off-target activities and, while clear-
ance is sufficiently low for mechanistic work, permeability is
poor and the compound has low oral bioavailability, limiting
its utility for in vivo studies.
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dynamic screens utilizing the site-specific phosphorylation of
acetyl CoA carboxylase (ACC) isoforms 1 and 2 by AMPK.
The assays either directly measured the ratio of pACC to total
ACC as a robust proximate target engagement readout for
AMPK activation; or measured suppression of de novo lipo-
genesis (DNL), or activation of fatty acid oxidation (FAO) as
functional consequences of increased ACC phosphoryla-
tion/inhibition.14 Following incubation with 3T3-L1 adipo-
cytes, Compound 3 suppressed DNL with an IC50 of 1.1 ꢀM
(Table 1).
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Our primary in vivo pharmacodynamic screen assessed tar-
get engagement (TE) (pACC/ACC elevations) in liver and
muscle in db/+ mice 2 h after a single oral dose. Tissue con-
centrations of compound were also collected and the combina-
tion of pACC elevation and exposure ratios in the two tissues
were used to infer the degree of liver-selectivity.
Compound 3 (30 mpk) caused robust increases in liver TE,
but did not elevate pACC/ACC in muscle in db/+ mice (Table
1). The 51-fold higher exposure in liver vs. muscle further
suggested that the compound preferentially distributes to the
liver.
Despite a relatively short plasma half-life and a free fraction
in plasma below the level of quantification, compound 3
served as a useful tool to interrogate the effects of β1-selective
AMPK activation in the liver. While reductions in plasma
insulin were evident following chronic administration in an
insulin resistant mouse model (diet-induced obese mouse),
compound 3 failed to cause reproducible glucose lowering in
rodent models of T2DM.15
Figure 2. Hit-to-lead Progression of the Series and Evolution
from Hepatic to Systemic β1-Selective Activators
As discussed, several studies have implicated activation of
AMPK as a principle mediator of many of the beneficial ef-
fects of exercise on glucose and lipid utilization.4,16 Our stud-
ies demonstrated that beneficial effects on lipids can be real-
ized with hepatic β1-selective activation in rodents, however
glucose homeostasis remains unaffected. Reports have sug-
gested that β2-containing AMPK complexes predominate in
skeletal muscle.17 This suggested that enhancing distribution
to muscle and increasing the degree of β2-containing AMPK
activation had the potential to provide greater efficacy in re-
solving hyperglycemia.
With the potential cardiac adverse effects in mind, we ini-
tially focused on identifying liver-selective molecules. Liver
AMPK activation is anticipated to provide some benefits, in
particular in addressing dyslipidemia and improving insulin
sensitization.12
After failing to identify any progressible leads from multiple
high throughput screens of the Merck compound collection,
we turned to a fragment screening approach. From a library of
ca. 25,000 compounds, compound 2 was identified as a weak
(EC50 ~30 ꢀM) activator of AMPK subtype 1 (α1β1γ1). A
rapid hit-to-lead effort identified compound 3 with significant-
ly improved potency at AMPK1 (α1β1γ1) and the other five
β1 containing AMPK complexes. However activation of the
β2-containing complexes remained low. Compound 3a is an
analog of 3 with very similar properties that became a standard
in our in vitro enzyme assays. To address any potential varia-
bility in the assay, enzyme activation data (both EC50 and
%Max) (Table 1) are reported relative to data for this com-
pound (supplemental). Since activity for β1 containing com-
plexes was usually comparable (and similarly activity for β2-
containing complexes was comparable), screening was typi-
cally run with one representative β1-containing enzyme
(AMPK1) and one representative β2- containing enzyme
[AMPK10 (α2β2γ1)]; data provided a gauge as to the relative
β1- vs. β2- complex selectivity for each analog. X-ray crystal-
lographic data for compound 3a bound to AMPK has been
reported.13
Nothing in the SAR we had generated thus far suggested a
means to improve β2 activation. With no relevant structural
information available to rationally modulate activity, the team
focused on addressing the ADME/PK challenges while look-
ing for SAR that might be used to drive improved pan-
activation.
We initially focused on two key goals: dialing-out liver se-
lectivity and enhancing the free fraction in plasma – some-
thing that we anticipated would be needed to avoid difficulties
in clinical development and translation.
In looking at the properties that might contribute to liver
distribution, we focused on two areas: susceptibility to liver-
uptake transporters and membrane permeability. Cell-based
studies confirmed that compound 3 is a substrate of OATP1B1
and 1B2. In addition, assessment in LLC-PK1 cells deter-
mined that permeability of the compound was relatively low
(Papp ~9 *10-6 cm/s). We therefore hypothesized that active
uptake into hepatocytes was more rapid than passive diffusion
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into/out of the cells resulting in compound sequestration in
liver.
Liver-selectivity was reduced in a series of aliphatic acids
represented by compound 4. Compound 4 was more β1-
selective than compound 3 with similar activity at AMPK1 but
reduced potency at AMPK10. However, cell activity was simi-
lar with an IC50 of 1.4 ꢀM for suppression of DNL. In vivo,
the compound caused significant elevation of liver and muscle
TE with comparable exposure in the two tissues (liver/muscle
concentration ratio ~4) (Table 1).
the two compounds diverged. Compound 4a maintained a
similar profile to compound 4 with significant TE in liver and
muscle and a low liver/muscle exposure ratio (~7). Exposure
of compound 4b was almost exclusively limited to liver (liv-
er/muscle concentration ratio ~649) with no increase in muscle
pACC despite robust TE in the liver (Table 1).
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Another approach to enhancing polarity is represented by
compound 5 where we attempted to add heteroatoms in the
carbocyclic acid moiety. This had a more limited impact on
PPB relative to the changes to the biphenyl and core regions,
but resulted in a similar in vivo distribution pattern to com-
pound 4b with liver-specific TE and a liver/muscle exposure
ratio of ~192. One serendipitous finding focused on the in
vitro selectivity profile of the compound. While activity at
AMPK1 was still comparable to the other analogs, compound
5 was the first in the series where we saw modest increases in
the maximal activation of β2 containing complex AMPK10
(Table 1).
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Scheme 1. Synthesis of MK-8722a
Figure 3. Evolution of Compounds with Improved Unbound
Fraction in Plasma and Identification of Systemic Pan-Activator
MK-8722
Similar to compound 3, compound 4 remained a substrate of
the OATP transporters. Permeability for the compound was
similar to compound 3 (Papp ~13 *10-6 cm/s) suggesting that
compound 4, while still a substrate, may have reduced suscep-
tibility for active uptake. Plasma protein binding for the com-
pound was little improved relative to compound 3 (Table 1) so
we turned our attention towards altering physicochemical
properties to address this.
aReagents and conditions: (a) NCS, AcOH, 80 °C, 18 h; (b) NaI, AcOH,
90 °C, 2 h; (c) SnCl2.2H2O, EtOH, 70 °C, 0.5 h; (d) thiophosgene,
DMAP, THF, 22 °C, 1 h; (e) KOH, MeI, EtOH, 22 °C, 0.5 h; (f) oxone,
MeCN/H2O, 22 °C, 18 h; (g) 4-biphenylboronic acid, K3PO4,
Pd(OAc)2, n-butyldiadamantylphosphine, THF/H2O, 45 °C, 18 h; (h)
Et3N, SEMCl, THF, 0-22°C, 15 min; (i) 1,4:3,6-dianhydro-2-O-[tert-
butyl(dimethyl)silyl]-D-mannitol, DBU, DMA, 22 °C, 18 h; (j) KHSO4,
HCO2H, 22 °C, 18 h then NaOH, THF 0-22 °C, 18 h.
Efforts to increase polarity focused on changes to the ben-
zimidazole core and the lipophilic biphenyl tail (Figure 3).
Replacement of the terminal phenyl ring with a pyrrolidine led
to the identification of compound 4a. cLogP was improved
relative to compound 4 (5.8 vs. 7.3) and we observed a com-
mensurate improvement in plasma protein binding with ~1%
free in 10% human serum. Introduction of a heteroatom in the
core led to compound 4b with further improvements in cLogP
(4.3). Again, these enhancements to polarity translated to im-
proved plasma protein binding with ~5.4% free in 10% human
serum. When we looked at 100% human serum, we were grati-
fied to find that the unbound fraction was indeed measurable
(~0.6%) (Table 1). Compounds 4a and 4b had comparable
activity to compound 3 at AMPK1 but degraded AMPK10
potency. Both compounds had lower cell potency (as meas-
ured by reduction of DNL in adipocytes) vs. compound 4 with
IC50s of 5.3 and 5.4 ꢀM, respectively. In vivo, the behavior of
Permeability for the analog with measurable free fraction in
plasma (compound 4b) was enhanced relative to earlier ana-
logs (Papp ~22*10-6 cm/s) suggesting that the degree of liver
selectivity again likely rested on rates of active transport into
the liver. The SAR raised the prospect that the physicochemi-
cal properties that we anticipated would drive to improved
PPB (increased polarity) were running counter to those that
would enhance systemic compound distribution. This suggest-
ed that we needed to overhaul our strategy if we wanted to
accomplish both goals. Since polar acid moieties serve as key
recognition elements for the OATP transporters, this became a
major focus of optimization efforts. Replacement of the acid
was also anticipated to reduce binding to albumin and increase
the unbound fraction of drug in plasma. In bile-duct cannulat-
ed rat studies, the primary routes of elimination of compound
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3 were biliary excretion of the parent molecule and parent
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compound 4, it was not possible to compare the free fraction
for the two compounds so we relied on target engagement
assays to confirm we had attained similar degrees of AMPK
activation. Rodent liver is almost exclusively made up of β1-
containing AMPK complexes.18 Given the similar AMPK1
potency and maximal activation for both compounds as well
as their comparable, balanced tissue distribution, we anticipat-
ed that liver pACC would provide a means to compare the
systemic target engagement attained by each compound. Both
compounds caused a similar elevation of liver pACC/ACC
(Fig 4A). However, the compounds diverged in their effects
on muscle pACC (Fig 4B-C). Muscle contains significant
amounts of β2-derived AMPK complexes.17,18 Compound 4
was able to elevate pACC/ACC in muscle at the higher 30
mpk dose – presumably due to robust activation of β1 contain-
ing AMPK complexes and a modest degree of β2-complex
AMPK activation. However MK-8722 increased pACC/ACC
to a much greater extent, reflecting the ability of the com-
pound to robustly activate both β1 and β2-containing com-
plexes. Importantly, the compounds also caused profoundly
different effects on glucose. Relative to vehicle treated con-
trols, compound 4 administration had no significant effect on
glucose at either dose (albeit with some variability in the high
dose animals). By contrast, MK-8722 induced a significant
reduction in glucose at both doses.
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glucuronidation followed by biliary excretion of the glucu-
ronide (data not shown). This provided additional rationale for
the strategy of replacing the acid moiety.
We capitalized on the slight improvement in β2 activation
observed for compound 5 by focusing on furan and pyran
based moieties and replaced the acid moiety with alcohols and
ethers. This led to a series of alcohol- and sugar-based analogs
that eventually identified MK-8722 (Figure 3).
The medicinal chemistry route for MK-8722 is outlined in
Scheme 1. The aza-benzimidazole ring was constructed from
commercially available 5-chloro-3-nitropyridin-2-amine via
halogenation of the 6-position, reduction of the nitro group
and cyclization with thiophosgene. Subsequent methylation
and oxidation to the sulfoxide afforded the fully functionalized
core. Palladium catalyzed coupling with biphenylboronic acid
and SEM protection prepared the way for substitution with the
protected sugar moiety. A final deprotection afforded MK-
8722.
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Figure 4. Effects of Compound 4 and MK-8722 in eDIO mice.
(A) Effects on tissue pACC/ACC. (B-C) Effects on fasting blood
glucose. N=8 in each group.
Figure 5. Effects of MK-8722 on Glucose and Xylose Excursion
During an oGTT in Lean C57BL/6 Mice. (A) Effects on the blood
glucose/time curve. (B) AUC of the glucose/time curve in A. (C)
Effects on the blood glucose/time curve. (D) Effects on the blood
xylose/time curves. N=8 in each group.
The acid-replacement strategy proved successful in allowing
moderation of both liver sequestration and plasma protein
binding while increasing activation of the β2-containing
AMPK subtypes. MK-8722 is a potent, systemic activator of
all twelve AMPK complexes (supplemental). The compound
has high permeability (Papp ~24 *10-6 cm/s), is not a substrate
of OATP1B1 or OATP1B2 and has measurable free fraction
in plasma (Table 1).
We subsequently assessed the effects of MK-8722 admin-
istration in an oral glucose tolerance test (oGTT) in eDIO mice
(Fig 5A-B). The compound dose-dependently lowered fasting
glucose as well as the glycemic excursion following the glu-
cose challenge. We confirmed that these effects were not due
to reduced glucose absorption by running a similar study
where xylose was co-administered with the glucose challenge.
While the effects on glucose were recapitulated (Fig 5C), there
was no change in xylose absorption relative to vehicle treated
controls (Fig 5D). Similar effects on fasting blood glucose and
glucose excursion during a GTT have been observed for the
compound across a range of normal, insulin resistant and
frankly diabetic rodent models.19 The minimum efficacious
exposure in eDIO mice was ~1 ꢀM (~1 nM unbound), alt-
hough more broadly across rodent models exposures of ~3 ꢀM
(3 nM unbound) are required for significant glucose lowering.
In order to assess the impact of increased β2 activity on gly-
cemic effects, we compared the effects of 10 mpk and 30 mpk
of MK-8722 and the systemic β1-selective compound 4 in
eDIO mice (Fig 4). These mice are hyperinsulinemic with
slightly elevated blood glucose levels, serving as a useful
model of insulin resistance/ pre-diabetes. The assay examined
acute compound effects on fasting blood glucose levels over
the course of 2 h. Compounds were administered in 0.25%
MC, 5% Tween-80, 0.02% SDS. Plasma exposure and both
liver and muscle pACC were assessed at the end of the study
(approximately 2-3 h post-dose). Both compounds achieved
high exposure in plasma (MK-8722: 18 and 59 ꢀM; compound
4: 43 and 124 ꢀM). Given the high plasma protein binding of
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8.
Arad, M.; Seidman, C. E.; Seidman, J. G. AMP-activated
In a screen of 115 enzymes, transporters and receptors, MK-
8722 had 3-10 ꢀM activity at 4 common off-targets [serotonin
5-hydroxytryptamine (5-HT2A), dopamine transporter (DAT),
norepinephrine transporter (NET), monoamineoxidase-A
(MOA-A) and monoamine oxidase-B (MOA-B)]. None of
these activities are anticipated to result in any significant
pharmacology at the (unbound) exposures relevant for AMPK-
mediated efficacy. Pharmacokinetics in preclinical species
were supportive of QD dosing in the clinic.19
protein kinase in the heart: role during health and disease. Circ. Res.
2007, 100, 474-88.
9.
adenosine 5'-monophosphate activated protein kinase (AMPK) modu-
lators and human diseases. J. Med. Chem. 2015, 58, 2-29.
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Rana, S.; Blowers, E. C.; Natarajan, A. Small molecule
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Cameron, K. O.; Kurumbail, R. G. Recent progress in the
identification of adenosine monophosphate activated protein kinase
(AMPK) activators. Bioorg. Med. Chem Lett. 2016, 26, 5139-5148.
11.
Wojtaszewski, J. F. A-769662 activates AMPK beta1-containing
complexes but induces glucose uptake through PI3-kinase-
dependent pathway in mouse skeletal muscle. Am. J. Physiol. Cell
Physiol. 2009, 297, C1041-52.
Treebak, J. T.; Birk, J. B.; Hansen, B. F.; Olsen, G. S.;
In summary, MK-8722 is a selective, small molecule, direct,
pan-AMPK activator. Our efforts to arrive at the molecule also
identified several tool compounds that have been useful in
assessing the in vitro and in vivo effects of liver-selective
and/or β1-selective AMPK activation. Based on our studies,
both β1- and liver-selective activators have robust effects on
lipid metabolism in rodents. While these may prove to be use-
ful for various disease conditions, they were not sufficient to
improve glucose homeostasis in rodents. Data obtained using
MK-8722 suggest that β2 activation is required to modulate
glycemic control, reinforcing the link between glucose uptake
following exercise and activation of β2 AMPK complexes.
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Viollet, B.; Guigas, B.; Leclerc, J.; Hebrard, S.; Lantier, L.;
Mounier, R.; Andreelli, F.; Foretz, M. AMP-activated protein kinase
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Xiao, B.; Sanders, M. J.; Carmena, D.; Bright, N. J.; Haire,
L. F.; Underwood, E.; Patel, B. R.; Heath, R. B.; Walker, P. A.; Hal-
len, S.; Giordanetto, F.; Martin, S. R.; Carling, D.; Gamblin, S. J.
Structural basis of AMPK regulation by small molecule activators.
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Fullerton, M. D.; Galic, S.; Marcinko, K.; Sikkema, S.;
Pulinilkunnil, T.; Chen, Z. P.; O'Neill, H. M.; Ford, R. J.; Palanivel,
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Supporting Information Available: syntheses of compounds 3-6, in
vitro enzyme activation data for compounds 3a and 6. The supporting
information is available free of charge on the ACS Publications web-
site at DOI: XXX
15.
Lan, P.; Romero, F. A.; Wodka, D.; Kassick, A. J.; Zhou,
G.; Chen, Y.; Zhang, X.; Zhang, A.; Ying, L.; Trujillo, M. E.; Shao,
Q.; Wu, M.; Xu, S.; He, H.; Chapman, K. T.; Weber, A.; Sebhat, I.
K.; Makara, G. M. Hit-to-lead optimization and discovery of 5-((5-
([1,1'-biphenyl]-4-yl)-6-chloro-1H-benzo[d]imidazol-2-yl)oxy)-2-
methylbenzoic acid (MK-3903): a novel class of benzimidazole-based
activators of AMP-activated protein kinase. J. Med. Chem. 2017, 60,
9040-9052.
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Table 1. Profile of Compounds 3-6: Fold Change in phosphorylated AMPK1 (pAMPK1) and pAMPK10 Activation vs.
Cmpd 3a, Effects on DNL in 3T3-L1 Adipocytes, cLogP, Unbound Fraction in Human Plasma, Fold Change in Muscle
pACC and Liver/Muscle Exposure Ratio in db/+ Mice
1
2
3
4
5
6
7
8
9
AMPK1 Fold
AMPK10
D
NL
IC50
(ꢀM)
Human Fu:
Liver
pACC/ACC
Muscle
pACC/ACC
Change
vs. Fold Change vs.
Cmpd 3a
Papp
Cliver
Cmus-
Cm
pd
(*10-6
/
10% serum
(100% serum)
Cmpd 3a
Fold Change Fold Change
cm/s) d
e
EC50a
EC50a
cle
vs. Vehiclee
vs. Vehiclee
d
a
(%Max)b
(%Max)b
3
4
1.8 (1.0)
7.5 (1.0)
0.52 (0.8)
18 (0.8)
11
9.4
0.2
0.3
1.7
1.4
1.1
2.0
51
4
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
1.
4
12.9
4a
4b
5
6.3 (0.9)
4.3 (1.0)
3.3 (1.0)
1.0 (0.5)
100 (0.7)
6.0 (1.1)
3.3 (1.5)
0.3 (1.9)
5.
3
NDc
21.5
NDc
23.6
1.0
1.8
2.0
1.5
1.9
2.3
1.5
1.2
2.9
7
5.
4
5.4 (0.6)
0.2
650
190
3
3.
9
6
N
4.6 (0.1)
Dc
aAverage of at least 2 experiments, each using a 10-point titration. Highest activation achieved at titrations to 50 ꢀM com-
pared to the maximal activation achieved for AMP. cNot determined. dAverage of at least 3 experiments. dN=7 animals in
a single experiment.
b
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