W. Hur et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5916–5919
5917
variety of cancers. However, a recent clinical phase II study re-
ported that CI-1033 is associated with severe toxicity, suggesting
that further development of the drug seems unlikely.5,6 Here, we
report that such irreversible HER-TK inhibitors are also capable
of potently inhibiting the Tec-family kinase Bmx/Etk. To our
knowledge, this is also the first report of a sub-micromolar
Bmx/Etk inhibitor.
As both the reversible and irreversible quinazoline inhibitors
have been reported to be highly selective to HER-TKs, we hypoth-
esized that other kinases bearing this unique cysteine residue
might also be targeted by these inhibitors. We first performed a
sequence alignment to determine which other protein kinases
possess an equivalently positioned cysteine to Cys773 of EGFR.
In addition to the other HER-TKs HER2 and HER4, eight more ki-
nases were identified, including Jak3, Blk, Lkb1, and the Tec-fam-
ily kinases (Bmx, Btk, Itk, Tec, and Txk)6 (Fig. 2a). In order to
determine which of these kinases might also be targeted by irre-
versible quinazoline inhibitors, we tested PD168393 and CI-1033
used to design cysteine alkylating inhibitors for Rsk,8 VEGFR,9
and Tec-family kinase BTK.6
We next used mass spectrometry to confirm the covalent nat-
ure of the inhibition, the stoichiometry of binding, and the site(s)
of modification of the inhibitors on recombinantly expressed
Bmx kinase domain. After incubation with PD168393, an average
mass of 31,732 Da was measured for the Bmx kinase domain. This
represents an increase of 369 Da, which is consistent with the
covalent addition of a single inhibitor molecule (Fig. 3a and b). A
second observed species corresponds to the covalent addition of
the singly phosphorylated form of the domain by a single inhibitor
molecule. Similar results were obtained upon incubation with CI-
1033, as the expected mass increase of 485 Da was observed for
both protein signals (Fig. 3c).
To determine the site(s) of modification, the inhibitor-modified
samples were reduced with dithiothreitol, alkylated with iodoacet-
amide, and subjected to chymotryptic digestion. Subsequent anal-
ysis by LC/MS/MS identified several overlapping species that
suggest Cys496 as the dominant nucleophilic site, which is analo-
gous to Cys773 of EGFR in the sequence alignment. Figure 3d and e
show the tandem MS spectra of the chymotryptic peptide ISNGCLL
modified with PD168393 or CI-1033, respectively. It should be
noted that partial modification of Cys516 by PD168393 (Supple-
mentary Fig. 1a) was also observed. However, the signal for this
species was more than 400 times lower than the signal for species
containing modified Cys496 (Supplementary Fig. 1b). Furthermore,
iodoacetamide-modified peptides of high signal intensity were
identified for every cysteine residue in the Bmx kinase domain ex-
cept for Cys496 (data not shown).
(Fig. 1) against
transformed Ba/F3 cell lines.7 PD168393 was also tested against
panel of sixty biochemical kinase assays. Of the reactive
a panel of 37 different Tel-tyrosine kinase
a
cysteine-containing kinases, the biochemical panel included EGFR,
HER2/4, Jak-3, Blk, Lkb-1, Bmx, BTK, and Itk, while the cellular
panel contained Jak3, Bmx, and Blk. While moderate to no inhibi-
tion was seen for the majority of the kinases tested, CI-1033 dem-
onstrated potent inhibitory activity against Bmx and Blk in both
biochemical and cellular assays, whereas PD168393 exhibited
only moderate potency against Bmx (Fig. 2b and Supplementary
Tables 1 and 2). The observation that only a subset of the kinases
possessing an equivalently positioned reactive cysteine are signif-
icantly inhibited by these compounds demonstrates that potent
inhibition requires a combination of non-covalent recognition of
the scaffold by the kinase and proper positioning of the reactive
cysteine nucleophile. Indeed, similar principles were recently
To confirm that Cys496 of Bmx is important for the cellular
activity of the inhibitors, we created a Ba/F3 cell line stably
transformed with a Tel-C496S Bmx mutant kinase domain. This
substitution resulted in almost 30-fold less sensitivity to
PD168393 (IC50 = 8.58 and 0.324
respectively, Fig. 4). We next examined the capability of the
compounds to inhibit full-length Bmx in cellular context.
lM for mutant and WT,
a
Flag-tagged Bmx was transfected to human prostate cancer
LNCaP cells, and an in vitro kinase assay was performed using
immunoprecipitated Flag-Bmx in the presence or absence of
reversible and irreversible clinical EGFR inhibitors (Fig. 5a).10
The in vitro autophosphorylation of Bmx, monitored by mea-
suring incorporation of labeled phosphate from 32P-ATP to the
immunoprecipitated Flag-Bmx, was almost completely inhibited
upon treatment with either PD168393 (10
(10 M), whereas it was not affected by treatment of the po-
tent reversible EGFR inhibitors gefitinib (10 M) and erlotinib
(10 M). The same result was obtained when Flag-Bmx was
lM) or CI-1033
l
l
l
immunopurified from the cells that were pre-treated with the
drugs for 4 h. Immunoblot analysis using anti-phosphotyrosine
antibody demonstrated only the irreversible but not the revers-
ible inhibitors, were able to inhibit phosphorylation (Fig. 5b).
These results demonstrate that both PD168393 and CI-1033 in-
hibit the activity of Bmx in cells in
fashion.
a Cys496-dependent
Unlike other Tec-family kinases that are preferentially
expressed in hematopoietic cells, Bmx/Etk is expressed in other
cell types as well, including endothelial, epithelial, and impor-
tantly metastatic carcinoma cells.11 As Bmx is involved in the
migration of both epithelial and endothelial cells,12 Bmx inhibi-
tors could potentially be used for antimetastasis and antiangio-
genesis therapies. Blk is a key player in signaling B-cell growth
inhibition in response to surface IgM cross-linking. Thus, Blk
inhibitors could also prevent B-cells from apoptosis13 and be
used to boost immune functions. Here we have demonstrated
that kinases such as Bmx and Blk can potently be inhibited by
covalent inhibitors from the 4-anilinoquinazoline scaffold class.
Figure 2. Irreversible quinazoline EGFR inhibitors cross-react with other tyrosine
kinases that possess a conserved reactive cysteine in their kinase domain. (a) The
sequence of EGFR active site and its alignment with ten other kinases. The common
putative nucleophilic cysteine residue is highlighted in bold. (b) Inhibition (IC50
,
lM) of kinases by PD168393 and CI-1033 examined by biochemical enzymatic
assays and cytotoxicity assays using Tel-tyrosine kinase transformed Ba/F3 cells
(ND, not determined).
Hur, Wooyoung
