crystal structure in a related kinase, this information
helped guide the optimization process and shed light on
key SAR. Both compounds show inhibition of P-p38a in
cells and are highly selective against other kinases in the
MAK pathway.
(tributylstannyl)isothiazole Pd(PPh3)4, PhMe, 120°C,
34%
Having identified two potent MKK3 inhibitors, their inhibi-
tory activity was tested against p38a phosphorylation in
cells (Figure 5). Human leukemic monocyte U937 cells
were treated with compounds 4 and 6 one hour prior to
TNFα stimulation; after which phospho-p38a (P-p38a)
was quantified using ELISA. Both compound 4 and
compound 6 showed dose dependent inhibitory activity
against p38a phosphorylation with IC50 values 2.7 uM
and 0.73 uM, respectively. The large shift between in
vitro inhibition and cell potency is likely a result of
MKK3’s Km for ATP (400-600nM)19.
The use of these compounds to help understand the role
of MKK in MAPK signaling, as well as the therapeutic
hypothesis behind MKK inhibition, will be reported in due
course.
1 Kumar, S.; Boehm, J.; Lee, J.C.; Nat Rev Drug Discov.,
2003, 9, 717-26.
2 Lee, J.C.; Kumar, S.; Griswold, D.E.; Underwood, D.C.;
Votta, B.J.; Adams, J.L.; Immunopharmacology, 2000, 47(2-
3),185-201.
3 Wagner, G.; Laufer, S.; Med Res Rev., 2006, 26 (1):1-62.
4 Zarubin, T.; Han, J.; Research, 2005, 15, 11–18.
5 Mjanov, N.; Kauffman, R.S.; Spencer-Green, G.T.; Arthritis
Rheum., 2009 60(5):1232-41.
6 Hen, S.B.; Cheng, T.T.; Chindalore, V.; Damjanov, N.;
Burgos-Vargas, R.; Delora, P.; Zimany, K.; Travers, H.;
Caulfield, J.P.; Arthritis Rheum., 2009, 60(2), 335-44.
Figure 5. Dose-dependent inhibitory activity of com-
pound 4 and compound 6 against p38a phosphorylation
in cells.
7
Simon, J.; Arthur, C.; Ley, S.; Nature Reviews Immunolo-
gy, 2013, 13,679–692.
8
Goldstein, D.M.; Gabriel, T.; Curr Top Med Chem., 2005;
5(10), 1017-29.
Characterization of the role of MKK3 and MKK6 in MAPK
signaling requires an in-depth understanding of the se-
lectivity profile across other kinases in the pathway.
Thus, compounds 4 and 6 were tested against a com-
prehensive panel of kinases found within the p38 signal-
ing pathway, including MKK6. As shown in Table 1,
both compounds were approximately 10x less potent
against the MKK6 isoform. In addition, compound 4 ex-
hibited >100-fold selectivity over all of the kinases in the
panel, whereas compound 6 showed moderate potency
against TAK1 kinase.
9
Hammaker, D.; Firestein, G.S.; Ann. Rheum. Dis., 2010,
69, i77-i82.
10
Hammaker, D.; Boyle, D.L.; Topoloewski, K.; Firestein,
G.S.; J Inflamm (Lond), 2014, 11: 14.
11
Yoshizawa, T.; Hammaker, D.; Boyle, D.L.; Corr, M.;
Flavell, R.; Davis, R.; Schett, G.; Firestein, G.S.; J
Immunol., 2009, 183(2),1360-7.
12 Raingeaud, J.; Whitmarsh, A.J.; Barrett, T.; Derijard, B.;
Davis, R.J.; Mol. Cell. Biol.,1996 ,16(3), 1247-1255.
13 Caliper Life Sciences, A Perkin Elmer Company. Hopkin-
14
ApIC50 = -log([inhibitor]/(Vo/V-1)) where Vo = enzyme
activity in the absence of inhibitor and V = enzyme activity
in the presence of inhibitor
MKK6 p38a
p38g JNK1 JNK2 ERK1
15 SPR work conducted under contract at BIOSesnsor Tools
LLC. See Supporting Information.
4
6
<9
12
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
16 Molecular Operating Environment (MOE) software, MOE,
Chemical Computing Group Inc.
17 Han, J.; Lee, J-D.; Jiang, Y.; Li, Z.; Feng, L.; Ulevitch,
R.J.; J Biol. Chem., 2012, 151 (5):541-549.
18 Smith, C.R.; Dougan, D.R.; Komandala, M.; Kanouni, T.;
Knight, B.; Lawson, J.D.; Sabat, M.; Taylor, E.R.; Vu, P.;
Wyrick, C.; J.Med.Chem., 2015, 58, 5437-5444.
19 Internal Data no shown
ERK2 MEK1 MEK2 TAK1 MLK3 ASK1
4
6
>100
>100
>100
86
>100
>100
>100
23
>100
>100
>100
>100
Table 1. Fold Selectivity of 4 and 6 against a panel of
MAPK signaling kinases (Invitrogen)
FBBD has long been established as a reliably efficient
means to identifying novel chemotypes that can be rap-
idly optimized using structural information. Herein we
described the rapid optimization of two highly potent and
efficient MKK3/6 inhibitors from compound 1. Although
the binding pose of the fragment was based on a co-
4