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B. Mavunkel et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2404–2408
5. Wolfe, J. P.; Buchwald, S. L. J. Org. Chem. 2000, 65, 1144.
6. Assays were performed with inhibitor or suitable control
solvent added 10 ll per well in a 96-well microtiter plate
(Corning, NY). CaMKIId was diluted in enzyme buffer
(50 mM PIPES, pH 7, 0.2 mg/ml BSA, 1 mM DTT) and
added 10 ll per well. Reactions were initiated with 30 ll
reaction buffer (62.5 mM PIPES, pH 7, 0.25 mg/ml BSA,
33.3 mM MgCl2, 83 lM ATP, 0.4 mM CaCl2, 8.3 lg/ml
calmodulin, 25 lM [His 5] Autocamtide-2, 120 nM [g-
33P]ATP) and incubated at RT for 3 min. Reactions were
terminated by transferring 25 ll to a UNIFILTER 96-well
P81microplate (Whatman, UK), pre-wet with 15 ll 1%
phosphoric acid. After 10 min, the plate was washed three
times with 1% phosphoric acid and one time with 95%
improvement in activity was noted. In fact, compound
15b demonstrated that we could overcome cell pene-
trance issues with sub-micromolar activity in our cell-
based assay. Furthermore, we validated the importance
of the extended basic group using morpholine or piper-
idine as piperazine variants. In both cases, enzyme activ-
ity was compromised. Finally, an unexpected benefit
resulted from relaxing the positioning of the extended
amine by replacing the piperazine with ethylene dia-
mine. While this compound showed no improvement
in enzyme activity, a 3-fold increase in cellular activity
was noted.
ethanol on
a
BiomekFX (Beckman–Coulter, CA)
In summary, novel pyrimidine-based inhibitors of CaM-
KIId were prepared. Homology model predictions were
used to advance the SAR from micromolar inhibitors to
low nanomolar inhibitors. Activity in cell-based assays
was improved by introducing basic groups at the phenyl
3-position and generating a predicted salt bridge with
Glu106. Our most potent inhibitor possessed a flexible
tether to the amine and exhibited an IC50 of 12 nM
against the isolated enzyme.
equipped with a vacuum manifold. Plates were dried for
approximately 60 min, scintillant was added to the wells,
and the plates were read on a TopCount NXT Microplate
Scintillation and Luminescence Counter (Perkin-Elmer,
MA).
7. An homology model of autoinhibited CaMKIId was built
based on the crystal structure 1A06 of autoinhibited rat
CaMKI. Because rat CaMKI shows high sequence
homology with CaMKIId, this model was used to study
inhibitors that were not ATP competitive. Due to the
lack of availability of a crystal structure of activated
CaMKIId, homology models were built based on crystal
structures 1CDK, 1PHK, and 1KOB. From these homol-
ogy models, we selected the one best explained the SAR.
The accuracy of this model was validated using point
mutation studies.
References and notes
1. Levy, D. E.; Wang, D-X.; Lu, Q.; Chen, Z.; Perumattam,
J.; Xu, Y-J.; Liclican, A.; Higaki, J.; Dong, H.; Laney, M.;
Mavunkel, B.; Dugar, S. Bioorg. Med. Chem. Lett. 2008,
18, 2390.
2. Levy, D. E.; Wang, D-X.; Lu, Q.; Chen, Z.; Perumattam,
J.; Xu, Y-J.; Higaki, J.; Dong, H.; Liclican, A.; Laney, M.;
Mavunkel, B.; Dugar, S. Bioorg. Med. Chem. Lett. 2008,
18, 2395.
3. Lu, Q.; Chen, Z.; Perumattam, J.; Wang, D-X.; Liang,
W.; Xu, Y-J.; Do, S.; Bonaga, L.; Higaki, J.; Dong, H.;
Liclican, A.; Sideris, S.; Laney, M.; Dugar, S.; Mavun-
kel, B.; Levy, D. E. Bioorg. Med. Chem. Lett. 2008, 18,
2399.
4. Zapf, A. In Transition Metals for Organic Synthesis:
Building Blocks and Fine Chemicals, 2nd ed.; Beller, M.,
Bolm, C., Eds.; Wiley-VCH: Weinheim, 2004; p 1344.
8. Amantini, D.; Beleggia, R.; Fringuelli, F.; Pizzo, F.;
Vaccaro, L. J. Org. Chem. 2004, 69, 2896.
9. HL-2 cells were pre-incubated with increasing concentra-
tions of an inhibitor for 1 h at 37 °C. CaMKII was
activated by cell treatment with 1 rmuM ionomycin for
15 min at 37 °C. Cells were then lysed in ice-cold M-PER
cell lysis buffer (Pierce) and frozen at À80 °C. Inhibitor
IC50 values were determined by an ELISA assay of
cell lysate measuring intracellular vimentin phosphory-
lated at Ser82 (Delling, Catalano, Wang, Higgins,
unpublished).
10. Solvation energy calculations were generated using ZAP
(Openeye Scientific Software).