Biphenyl amide p38 kinase inhibitors 3: Improvement of cellular and in vivo activity

Article abstract of DOI:10.1016/j.bmcl.2008.06.048

The biphenyl amides (BPAs) are a novel series of p38α MAP kinase inhibitor. The optimisation of the series to give compounds that are potent in an in vivo disease model is discussed. SAR is presented and rationalised with reference to the crystallographic binding mode.

Full text of DOI:10.1016/j.bmcl.2008.06.048

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4428–4432
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Biphenyl amide p38 kinase inhibitors 3: Improvement of cellular and
in vivo activity
Richard Angell , Nicola M. Aston, Paul Bamborough, Jacky B. Buckton, Stuart Cockerill , Suzanne J. deBoeck,
à
*
Chris D. Edwards, Duncan S. Holmes, Katherine L. Jones , Dramane I. Laine, Shila Patel , Penny A. Smee,
Kathryn J. Smith, Don O. Somers, Ann L. Walker
GlaxoSmithKline R&D, Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
The biphenyl amides (BPAs) are a novel series of p38a MAP kinase inhibitor. The optimisation of the ser-
Received 9 May 2008
Revised 10 June 2008
Accepted 10 June 2008
Available online 18 June 2008
ies to give compounds that are potent in an in vivo disease model is discussed. SAR is presented and
rationalised with reference to the crystallographic binding mode.
Published by Elsevier Ltd.
Keywords:
p38 Kinase
p38 Alpha
p38
CSBP
MAP kinase
Inhibitors
Biphenyl amide
BPA
Biphenyl amides
BPAs
Protein kinase X-ray structure
Binding mode
Structure-based drug design
Kinase selectivity
Structure–activity relationships
PG-PS model
CIA model
The biphenyl amides are a novel series of p38 MAP kinase inhib-
itor. Initial lead optimisation focused on optimisation of the amide
substituent and of the 1,3,4-oxadiazole moiety.1 Replacement of
the oxadiazole by other heterocycles led, in most cases, to lower
bonds to the kinase backbone at Asp168 and Phe169 in the DFG-
motif.2 The oxadiazole oxygen was positioned near the acid of
the Glu71 sidechain. Modelling suggested that the isosteric
replacement of the oxadiazole by an amide would maintain the
hydrogen bond to Asp168 from the carbonyl oxygen, and in addi-
tion donate a hydrogen bond to Glu71 through the amide NH.
The amide functionality also offered potential for increasing
p38
a
activity. However, the 40-amide readily accommodated a
wide range of different groups which provided improved enzyme
and cellular potency and led to the discovery of 1.
X-ray crystallography of 1 bound to the ATP-site of p38
a
p38a potency by optimisation of the interactions with the lipo-
showed that the oxadiazole occupied a mainly lipophilic pocket,
philic pocket.
and that the nitrogen atoms on the oxadiazole formed hydrogen-
The synthesis of the compounds concentrated on two coupling
reactions. The first connected amines to an acid core, to form benz-
amides A, while the second coupled acids to an amine core to give
anilides B (Fig. 1).
Substituents were chosen with two goals in mind. The first was
to probe the pocket filled by the oxadiazole in the X-ray complex of
1 using a range of similarly sized groups. The second was to try to
* Corresponding author. Tel.: +44 1438 762914.
E-mail address: katherine.l.jones@gsk.com (K.L. Jones).
Present address: Arrow Therapeutics, Britannia House, 7 Trinity Street, London
SE1 1DA, UK.
à
Present address: University of Cambridge, Department of Biochemistry, Sanger
access the ‘‘DFG-out” conformation of p38a, first described in the
Building, Old Addenbrookes Site, 80 Tennis Court Road, Cambridge CB2 1GA, UK.
0960-894X/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2008.06.048

R. Angell et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4428–4432
4429
Me
Me
Glu71 O
O
O
Me
4'
Br
I
O
R1
N
H
3
Cl
H
N
OH
N
H
R2
A
O
O
a
b
O
1
N
Asp168 NH
Me
N
Asp168 NH
O
O
O
Me
Br
+
B
O
N
H
Glu71 O
4
Phe169 NH
OH
R1
N
H
5'
NH
O
c
O
O
B
O
R2
N
H
Asp168 NH
Me
N
H
d
Me
Figure 1. Benzamides (A) and anilides (B) showing ring numbering and p38
interactions.
O
OH
literature for the complex of BIRB-796.3 Discussion here will be
limited to biphenyl amides with the DFG-in binding mode: com-
pounds with the DFG-out binding mode will be reported
elsewhere.4
O
NH
R
Scheme 1. Reagents: (a) cyclopropylmethylamine, NEt3, THF, 74%; (b) bis(pinaco-
lato)diboron, PdCl2(dppf), KOAc, DMF 54%; (c) Pd(PPh3)4, 1 M Na2CO3 (aq), DME,
78%; (d) amide coupling, for example amine, HATU, DIPEA, DMF. (For 4 the order of
amide formations was reversed.)
Appropriately substituted, both benzamides and anilides gave
rise to potent inhibitors. Table 1 shows p38
a activity data for a
range of benzamides, prepared as in Scheme 1.
The cyclopropyl amide 3 (Ki 12 nM) is one of the most potent
inhibitors from the benzamide series. The X-ray structure of 3
may be as a result of a clash with Leu74 or Phe169 in the pocket
floor. Groups such as isobutyl, cyclopentyl and cyclohexyl (6–8)
are weakly active. Although they would penetrate the pocket to a
similar depth to 3, they are wider and may clash with Glu71,
Leu75 or Leu171 in the pocket walls. Groups of similar size to
cyclopropyl such as isopropyl and cyclobutyl (9, 10) are over 10-
fold weaker than 3, which further indicates the tight steric
requirements.
Aromatic groups (11–13) also show size-dependent trends. The
smaller 5-membered heteroaromatics (12, 13) are more potent
than phenyl (11).
Anilides B (Fig. 1) were also synthesized (Scheme 2). X-ray com-
plexes of several examples have been solved, and are very like the
structure of 3 (data not shown). Both of the hydrogen-bonding
interactions made between the amide linker and the protein are
maintained, even though its direction is reversed. There is a slight
shift in the position of the attached substituent resulting in differ-
ent SAR for the two series of amides (Table 2).
complexed to p38a was solved and is shown (Fig. 2) superimposed
on that of 1 which has already been described.2,6 In the hinge re-
gion, the 40-amides of 1 and 3 make the same hydrogen bond to
Met109, and the substituents occupy the same region of the active
site.
The two toluene rings overlay very closely. As expected, the
oxygen of the 3-amide overlays closely with the N3 atom of the
oxadiazole. Both accept hydrogen bonds from the backbone NH
of Asp168. Compound 3 also donates an additional hydrogen bond
from the 3-amide NH to the sidechain of Glu71.
The cyclopropyl group fits tightly in a lipophilic pocket, whose
base is formed by the sidechains of Leu74 and Phe169, subtly rear-
ranged from their positions in the complex with 1. The sidechains
of Glu71, Leu75 and Leu171 form the pocket walls. Small groups,
such as the ethyl analogue (2), are less potent and would not fill
this lipophilic pocket completely. Cyclopropyl has the greatest
activity (3) which would indicate that it is a more optimal size.
Longer aliphatic groups (e.g. 4, 5) show decreasing activity which
As with the benzamides, activity against p38a of compounds
with the anilide alkyl substituents increases with size until the
optimal size is reached, then rapidly decreases (14–22). Alkyl
amides are weaker in the anilide series than in the benzamides.
While the cyclopropyl substituted benzamide (3) is one of the most
active compounds in that series, the cyclopropyl substituted ani-
lide (16) is not particularly potent. Propyl (17) is the most active
alkyl amide but is still threefold lower in activity than 3. The ani-
lides show a clear preference for aromatic substituents (23–28)
which are more potent than their analogues in the benzamide ser-
ies (compare 23 to 11). Despite the good potency achieved in this
series, the benzamides were preferred due to the potential toxicity
risks associated with aniline formation during metabolism of the
anilides.7
Table 1
Activities against p38
a
of small benzamide analogues (nM)5
Me
Me
O
O
N
H
N
H
H
N
O
R
O
N
1
Me
N
Compound
R
IC50
Ki
480
100
12
150
90
1600
430
1400
400
130
70
1
2
3
4
5
6
7
8
Not applicable
Ethyl
Cyclopropyl
Propyl
3000
610
75
970
550
Variation at the 40 amide position (29–37), Table 3, was intro-
duced into the benzamide series (Scheme 3). The 40-amide substi-
tuent points towards solvent in a similar way to that of the 40-
amide of oxadiazole 1 and the SAR trends at this position in the
amide series are very like those seen for the oxadiazoles.1 A wide
range of substitution on the 40-amide is tolerated. Aryl and benzyl
groups (29–33) show the greatest activity. Compounds from Table
Cyclopropylmethyl
Isobutyl
Cyclopentyl
Cyclohexyl
Isopropyl
Cyclobutyl
11000
2700
8800
2500
850
9
10
11
12
13
Phenyl
1,3-Thiazol-2-yl
1,3,4-Thiadiazol-2-yl
460
86
150
14
20
3
were typically 50-fold more potent than the equivalent
oxadiazoles.1

4430
R. Angell et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4428–4432
Figure 2. X-ray structure of 3 compared to 1 showing the 3-amide superimposed on the oxadiazole.
Table 3
Activities against p38
Me
Me
a
of small benzamide analogues (nM)5
a
O
Br
Me
MeO
O
NHBOC
NH2
R
N
H
b
H
N
O
Me
Me
c
O
N
O
R1
R1
N
Compound
R
IC50
Ki
12
5
7
4
2
O
R2
R2
N
H
NH2
3
Cyclopropylmethyl
3-Cyanophenyl
4-Methoxyphenyl
3-Methoxybenzyl
3-[(Methylsulfonyl) amino]benzyl
4-(N-Me)piperazinyl benzyl
NH2
Propanol
Propylmorpholine
Dimethyl propylamine
75
33
42
23
10
R3
29
30
31
32
33
34
35
36
37
Scheme 2. Reagents: (a) i—di-tert-butyl dicarbonate, NEt3, DCM, 63%, ii—(4-
methyoxycarbonylphenyl)boronic acid, Pd(PPh3)4, CsCO3, DME, 97%; (b) i—NaOH,
MeOH 49%, ii—HNR1R2, HATU, DIPEA, THF, iii—TFA, DCM; (c) R3CO2H, CDI, THF/
DMF.
21
3
610
270
650
230
97
43
100
370
Table 2
Activities against p38
a
of small anilide analogues (nM)5
Me
Me
O
Me
a
I
N
H
I
H
NH
N
O
OH
O
R
O
b
Compound
R
IC50
4000
Ki
630
170
350
40
270
70
330
1100
1900
12
Me
Me
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
Methyl
Ethyl
c
O
O
1100
2300
240
1700
450
2100
7000
12000
76
160
96
200
34
Cyclopropyl
Propyl
Cyclobutyl
Cyclopropylmethyl
Isobutyl
Cyclopentyl
Cyclohexyl
Phenyl
2-Furan
3-Furan
2-Thiophene
3-Thiophene
5-Isoxazole
R
N
H
MeO
H
H
N
N
O
O
Scheme 3. Reagents: (a) cyclopropylamine, HATU, HOBT, DIPEA, DMF; (b) (4-
methoxycarbonylphenyl)boronic acid, Pd(PPh3)4, Cs2CO3, DME; (c) i—NaOH, MeOH,
H2O, ii—various amide coupling conditions, for example amine, HATU, DIPEA, DMF.
25
15
32
5
The methyl on the toluene ring is one of the key requirements
for p38
a activity in the series. As reported for the oxadiazole BPAs,
40
6
modification in this region (38–45, Scheme 4) usually led to loss of

R. Angell et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4428–4432
4431
Table 5
H
X
X
Pharmacokinetic parameters of 3 measured in rat12
O
N
R
R
a
IV plasma clearance (ml/min/kg)
IV steady state volume of distribution (l/kg)
IV plasma terminal t1/2 (h)
Oral bioavailability
6
0.6
1.3
113%
0.1–0.3
c
R''
R'
O
R' = COOH, NH2
X = Br, I
R
N
Brain:plasma ratio
H
R'' =
R''
H
O
N
against AKT1, Aurora A, EGFR, EphB4, GSK-3b, JNK3, Lck, ROCK1
and Syk. A similarly selective profile was observed for other mem-
bers of the series. In addition, 3 was submitted to the KinomeScan
assay panel, which measures binding against over 200 kinases or
kinase domains fused to T7 bacteriophage.9a Compound 3 only
showed significant ability to displace an immobilised ATP-site li-
H
O
O
O
N
H
O
N
B
b
O
gand from p38a and p38b. This compares favourably to literature
data for p38 inhibitors VX-745 and BIRB-796 which in a smaller
panel were reported to bind to a number of off-target kinases.9b
Compounds were profiled in a series of assays measuring the
inhibition of a panel of p450 enzymes.10 Compound 3 was not a po-
Br
Scheme 4. Reagents: (a) HATU, HOBT, diisopropylethylamine, DMF, cyclopropyl-
amine or 3-furoic acid; (b) bis(pinacolato)diboron, potassium acetate, PdCl2(dppf),
DMF; (c) Pd(PPh3)4, 2 M Na2CO3 (aq), DMF. (For 38 the boronic ester and halide
were on opposite Suzuki coupling partners.)
tent inhibitor of isoforms 1A2, 2C19, 2D6 or 3A4 (IC50 > 10
it did inhibit p450 2C9 with IC50 of 4.4 M.
Along with other potent examples from the series, 3 was pro-
filed in cellular assays. It was a very potent inhibitor of TNF- re-
lease from peripheral blood mononuclear cells with IC50 of
250 nM, a considerable improvement over 1 (2.5
M).11 In addi-
tion, 3 reduced the level of TNF in response to LPS-stimulation
of whole blood with IC50 = 1
M.11
lM), but
l
activity as shown in Table 4.2 Only chlorine was able to adequately
replace methyl. When the methyl was replaced by smaller groups
such as hydrogen or fluorine (38, 41,42) the p38
a
a activity de-
l
creased by about 100-fold. Slightly larger groups, such as methoxy
(40, 44), caused a similar loss of potency. This data can be rationa-
lised by studying the X-ray structure of 3. The methyl appears to be
the optimal size to fill the small lipophilic pocket formed by Ala51
and Thr106. Wedged against the beta sheet in the N-terminal lobe,
this part of the site has little freedom to move in response to
changes in the ligand. Compounds from Table 3 were typically
50-fold more potent than the equivalent oxadiazoles. This
improvement is consistent with that seen for compound 3 over
1, and can be rationalised in the same way.
Compounds from the bis-amide series had an improved profile
over the oxadiazole series.1 Compound 3 had a Ki of 12 nM,5 a sig-
nificant improvement over the value of 480 nM for 1. Compound 3
also inhibited the phosphorylation of ATF-2 by p38
10 nM.8 Selectivity was assessed by screening against a panel of
56 protein kinases. Only p38 and p38b (Ki = 24 nM) were signifi-
a
l
Compound 3 had good pharmacokinetic properties in the rat
(Table 5).12 Oral bioavailability was excellent, with low clearance
and a moderate volume of distribution. The compound also
showed a low brain:plasma ratio, which would reduce any risk of
unwanted activity in the CNS.
Interestingly, the cyclopropylamide amide replacement (3) for
the methyl-oxadiazole (1) seemed to boost further the oral bio-
availability of 3, despite the additional hydrogen bond donor (1
showed oral bioavailability of 50% and similar clearance to 3).1
However, the substitution did not affect the brain penetration in
the rat, which was low for both compounds (brain:plasma ratio
for 1 was 0.3). A potential explanation for these findings is that
the complete oral bioavailability of 3 may have been driven by a
slightly improved solubility over 1, or may have been determined
by a different impact of active component of intestinal absorption,
rather than by simple passive transcellular absorption.
a with a Ki of
a
cantly inhibited by 3. The compound was at least 100-fold selective
Table 4
In addition, compound 3 showed excellent activity in a rat PG-
PS (peptidoglycan-polysaccharide) reactivation model, with
Activities against p38a
of methyl replacements (nM)5
ED50 = 0.02 mg/kg.2
This
compared
favourably
with
1
O
O
N
H
R1
N
H
R2
(ED50 = 15 mg/kg) and prednisolone (ED50 = 1 mg/kg). It was also
tested in a mouse collagen induced arthritis (CIA) model of rheu-
matoid arthritis,13 in which it totally prevented the progression
of arthritis at 15 mg/kg/day. At the higher dose of 30 mg/kg/day,
it had a beneficial effect on arthritis and reduced clinical score
compared to the level at the start of the dosing period. In this mod-
el 3 compared favourably with Enbrel, soluble recombinant TNF
HN
O
O
NH
receptor, dosed at 300 lg ip on alternate days, which reduced
inflammation but did not completely prevent disease progression.
O
R1
IC50
Ki
In conclusion, replacement of the oxadiazole ring of the early
Compound
biphenyl amide p38a inhibitors led to a series of compounds with
3
Me
H
Cl
Methoxy
Fluoro
R2
75
>16,000
160
3300
2900
12
>2500
25
520
460
greatly improved properties, including excellent cellular potency,
pharmacokinetic properties and oral activity. Future publications
will describe the continuing optimisation of the series to yield can-
didate quality molecules.
38
39
40
41
25
42
43
44
45
Me
H
Cl
96
5900
63
>16,000
>16,000
15
940
10
>2500
>2500
Acknowledgements
Methoxy
Dimethylamine
The authors thank Rick Williamson and Giovanni Vitulli for sug-
gestions and for contributing experimental details, Tony Cooper,

4432
R. Angell et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4428–4432
10 lM and considered active if <10% of binding to immobilised probes
remained compared to DMSO control.; (b) Goldstein, David M.; Gabriel,
Tobias Curr. Topics Med. Chem. 2005, 5, 1017 .
Rachel Hosking and Steve Flack for synthesis of compounds, Vipul
Patel for helpful discussions, and all other members of the p38
team for their many contributions.
10. The inhibition of recombinant CYP450 mediated O-dealkylase metabolism of
pro-fluorescent probe substrates were assessed in a fluorimetric assay with the
following recombinant human cytochrome P450s co-expressed in Escherichia
coli with human NADPH reductase (Bactosomes, CYPEX, UK); CYP2C9 with the
substrate 7-methoxy-4-trifluoromethylcoumarin-3-acetic acid (FCA); CYP1A2
with the substrate ethoxyresorufin (ER); CYP2D6 with the substrate 4-
methylaminomethyl-7-methoyxycoumarin (MMMC); CYP2C19 with the
substrate 3-butyryl-7-methoxycoumarin (BMC); and CYP3A4 with the
substrates 7-benzloxyquinolone (7BQ) and diethoxyfluorescein (DEF).
Compounds were prepared as 5 mM MeOH stock solutions with final
incubations containing <2% MeOH. The concentration range for the IC50
References and notes
1. Angell, R. M.; Angell, T. D.; Bamborough, P.; Brown, D.; Brown, M.; Buckton, J.
B.; Cockerill, S. G.; Edwards, C. D.; Jones, K. L.; Longstaff, T.; Smee, P. A.; Smith,
K. J.; Somers, D. O.; Walker, A. L.; Willson, M. Bioorg. Med. Chem. Lett. 2008, 18,
324.
2. Angell, R. M.; Bamborough, P.; Cleasby, A.; Cockerill, S.; Jones, K. L.; Mooney, C.
J.; Somers, D. O.; Walker, A. L. Bioorg. Med. Chem. Lett. 2008, 18, 318.
3. Pargellis, C.; Tong, L.; Churchill, L.; Cirillo, P.; Gilmore, T.; Graham, A.; Grob, P.;
Hickey, E.; Moss, N.; Pav, S.; Regan, J. Nat. Struct. Biol. 2002, 9, 268.
4. Angell, R.; Aston, N.M.; Bamborough, P.; Buckton, J.B.; Cockerill, S., de Boeck,
S.J.; Edwards, C.D.; Holmes, D.S.; Jones, K.L.; Laine, D.I.; Patel, S.; Smee, P.A.;
Smith, K.J.; Somers, D.O.; Walker, A.L.; Bioorg. Med. Chem. Lett., accepted for
publication.
determination assay was 0.1–100
0.2 mM NADPH regeneration system.
11. (a) Inhibition of LPS-stimulated TNF
peripheral blood mononuclear cells as described in Ref. 1. (b) Inhibition of
TNF release in response to LPS-stimulation was carried out in whole blood.
Heparinised blood drawn from normal volunteers was dispensed (100 l) into
microtitre plate wells containing 0.5 or 1.0 l of an appropriately diluted
compound solution. After 1 h incubation at 37 °C, 5% CO2, 25 l LPS solution in
RPMI 1640 (containing 1% -glutamine and 1% penicillin/streptomycin) was
added (50 ng/ml final). The samples were incubated at 37 °C, 5% CO2 for 20 h,
100–150 l physiological saline (0.138% NaCl) was added and diluted plasma
was collected using a Platemate or Biomek FX liquid handling robot after
centrifugation at 1300g for 10 min. Plasma TNF content was determined by
lM (spread over 9 points) and included a
a
release was measured in isolated
a
l
5. Enzyme IC50 and Ki determination, using an assay measuring displacement of a
fluorescent ATP-competitive inhibitor was carried out as described in reference
1.
l
l
L
6. An apo crystal of unphosphorylated human p38
a (expressed, purified and
crystallized as previously described) was soaked with 0.25 mM 3 for 1 day and
cryoprotected as for compounds 3 and 4 of the previous publication.2 X-ray
diffraction data were collected from the crystal at 100 K (using an Oxford
Cryostream) on a Rigaku-MSC RuH2R rotating anode X-ray generator with a
RAXIS IV++ image-plate detector. The data were processed with the HKL
package (Otwinowski, Z.; Minor, W. Methods Enzymol. 1997, 276:Macromol.
Crys. A, 307–326) and CCP4 program suite (Bailey, S. Acta Crystallogr., Sect. D,
1994, 50, 760–763). The structure was solved using the native p38 coordinates
(PDB entry 1WFC) as the initial model in refinement by REFMAC (Murshudov,
G.; Vagin, A.; Dodson, E. Acta Crystallogr., Sect. D, 1997, D53, 240–255). The final
R-factor achieved for the complex was 17.4%. Coordinates have been deposited
in the PDB as entry 3D7Z.
l
a
enzyme linked immunosorbant assay (ELISA) or using
technology (Luminex).
a multiplex bead
12. Pharmacokinetic parameters in male Lewis rats were determined following
intravenous (iv) and oral (po) administration at 0.5 mg/kg and 1.5 mg/kg,
respectively. Compound was administered as a solution in 10% DMSO: 45%
SBE Cyclodextrin, 45% MSA (IV) or 5% DMSO, 40% vitE, 40% PEG200, 15%
Mannitol (po). Blood was collected over a 24-h time period. Plasma was
prepared following centrifugation and compound extracted from 50
plasma using protein precipitation with acetonitrile. Samples were then
evaporated under nitrogen and re-suspended in 100 of 10:90
ll
7. (a) Harrison, J. H., Jr.; Hollow, D. J. J. Pharmacol. Exp. Ther. 1986, 238, 1045; (b)
Osazaki, Y.; Yamashita, K.; Ishii, H.; Sudi, M.; Tsuchitani, M. J. Appl. Toxicol.
2003, 23, 315.
ll
acetonitrile/water. Analysis was performed using LC–MSMS on the API365
with a 5 min fast gradient comprising 0.1% formic acid in water and 0.1%
8. Recombinant His-tagged human p38
p38 incubated in 200 mM Hepes pH 7.4, 625 mM NaCl; 1 mM DTT with 27 nM
active MKK6 (Upstate). Activity of the activated p38 was assessed using a
a was activated using 3 lM unactivated
formic acid in acetonitrile (mobile phases), 20
rate 4 ml/min and ODS3 Prodigy column (5 cm ꢀ 2.1 mm,
Pharmacokinetic data was generated using non-compartmental
l
l
injection volume, flow
a
5
l
m).
a
a
time-resolved fluorescence energy transfer (TR-FRET) assay. Biotinylated GST-
ATF2 (residues 19–96, 400 nM final), ATP (125 mM final) and MgCl2 (5 mM
final) in assay buffer (40 mM HEPES pH 7.4, 1 mM DTT) were added to wells
approach. The brain penetration was assessed in the Lewis rat, collecting
brains and terminal blood samples five minutes after an intravenous
administration at 1 mg/kg, using the same formulation as in the
intravenous PK study. Brains were homogenised in a water:methanol 1:1
containing 1
final) in NUNC 384-well black plate. The reaction was initiated by addition of
p38 (100 pM final) to give a total volume of 30 l. After 120 min incubation
(rt), 15 l of 100 mM EDTA pH 7.4 was added followed by detection reagent
(15 l) in buffer (100 mM HEPES pH 7.4, 150 mM NaCl, 0.1% w/v/BSA, 1 mM
ll of various concentrations of compound or DMSO vehicle (3%
solution and the homogenates analysed with
method similar to the one detailed for plasma analysis.
13. CIA is widely used animal model of arthritis in which the anti-
a protein precipitation
a
l
l
a
l
inflammatory and anti-rheumatic efficacy of drugs and novel compounds
predicted to have activity in RA are evaluated. This model is one of the more
widely used animal models of RA and shares many similarities with the
human disease, for example synovial hyperplasia, infiltration of
inflammatory cells, erosion of cartilage and bone and involvement of both
B and T lymphocytes. It has extensively been reported in the literature that
CIA can be inhibited by anti-cytokine reagents (IL-1b, IL-6 and to a lesser
DTT) containing anti-phosphothreonine-ATF2-71 polyclonal antibody (Cell
Signalling Technology, Beverly, Massachusetts, MA) labelled with W-1024 Eu
chelate (Wallac OY, Turku, Finland), and APC-labelled streptavidin (Prozyme,
San Leandro, CA). After 60 min further incubation (rt) the ATF-2
phosphorylation was measured using
a Packard Discovery plate reader
(Perkin-Elmer, Pangbourne, UK) as a ratio of specific 665 nm energy transfer
signal to reference Eu 620 nm signal. To calculate Ki from determined IC50 the
Cheng-Prusoff equation was used (Ref. 1). Ki = IC50/(1 + ([S]/Km)).
extent TNF
type II collagen + Freund’s Complete adjuvant injected intradermally at the
base of the tail followed weeks later by an intraperitoneal booster
injection of bovine type II collagen. The activity of compound in this
a). CIA was induced by sensitising DBA/1 mice against bovine
9. Assays were carried out by Ambit Biosciences, San Diego. The ability of
compounds to compete with the binding of the human kinase (expressed as
fusion to T7 bacteriophage) to immobilized ATP-site probe ligands was
determined as previously described, see: (a) Fabian, M. A.; Biggs, W. H., III;
Treiber, D. K.; Atteridge, C. E.; Azimioara, M. D.; Benedetti, M. G.; Carter, T. A.;
Ciceri, P.; Edeen, P. T.; Floyd, M.; Ford, J. M.; Galvin, M.; Gerlach, J. L.; Grotzfeld,
R. M.; Herrgard, S.; Insko, D. E.; Insko, M. A.; Lai, A. G.; Lélias, J.-M.; Mehta, S. A.;
Milanov, Z. V.; Velasco, A. M.; Wodicka, L. M.; Patel, H. K.; Zarrinkar, P. P.;
Lockhart, D. J. Nat. Biotechnol. 2005, 23, 329. Compounds were screened at
3
3
model was assessed by orally dosing mice twice daily for 14 days with 15 or
30 mg/kg starting when the mice were showing early signs of inflammation
in the paws. Enbrel (soluble TNF
a receptor) was tested in the same study as
a comparator. Clinical scores were monitored throughout the dosing period
as a measure of anti-inflammatory effect and at the end of the study, ankle
joints were processed histologically to assess effects on structural damage.