ACS Medicinal Chemistry Letters
Letter
thiol functionality (2) (Chart 1). Compound 3 was synthesized
as a control absent a reactive thiol. As the electrophile
present could we detect a mass for the Michael adduct (4). We
next synthesized 4 and found it to have improved binding
potency compared to 3 and EL-1148, the individual starting
fragments (Chart 2). Because 2 is an irreversible inhibitor of
Chart 1. Structure of Promiscuous Kinase Inhibitors 1−3
Chart 2. Structures of Bivalent Kinase Inhibitors
component, acrylamides are attractive because they have low
1
6,17
intrinsic reactivity.
Their relatively low reactivity will ensure
that the screen will selectively identify enzyme-templated
reactions. Conversely, electrophiles with high intrinsic reactivity
(
e.g., vinylsulfonamides) could produce conjugates in the
17
absence of enzyme.
We chose c-Src, a nonreceptor tyrosine kinase, as the initial
target to develop our methodology. c-Src has been extensively
studied over the years and implicated in a variety of diseases
1
8,19
including the metastasis of several cancers,
and few
wild-type c-Src, we compared compound 3, where the thiol of 2
5,8
selective inhibitors are available to study c-Src biology.
is replaced with a carbon. Compound 3 (K = 0.40 ± 0.02 μM)
i
Importantly, compound 1 is a competent inhibitor of c-Src (Ki
was found to be less potent than bivalent inhibitor 4 (K = 0.09
i
=
0.36 ± 0.08 μM). To ensure that our assay would identify
±
0.02 μM) for wild-type c-Src. To enable a comparison of
thiol 2 with bivalent inhibitor 4, we tested both for inhibition of
M c-Src and found 4 to be a more potent inhibitor of this
construct (2, 3M c-Src K = 0.20 ± 0.06 μM; 4, 3M c-Src K =
only acrylamides that react with 2 and not c-Src itself, we made
a mutant c-Src kinase with three nonessential, solvent-accessible
cysteines mutated to serines on the surface of c-Src (“3M” c-
Src: C277S, C483S, S496S). Of note, we found that compound
3
i
i
0
.12 ± 0.02 μM).
EL-1148 is a very weak inhibitor of c-Src (3M c-Src, IC50
70 ± 110 μM) but was readily identified in our method. In
2
is a time-dependent, irreversible inhibitor of wild-type c-Src,
=
but not of 3M c-Src.
4
A library of 110 acrylamide fragments (average molecular
weight = 235 Da, see Supplemental Table S1 for structures)
was incubated with 3M c-Src in the presence and absence of 2
traditional fragment-based screening, it could be difficult to
identify weak binding fragments using activity-based assays and
even more difficult to discriminate between ATP-competitive
and non-ATP-competitive fragments. Using biochemical
activity assays, we further demonstrated that EL-1148 is
noncompetitive with ATP, suggesting that it is binding in a
pocket adjacent to the ATP-pocket (Supplemental Figure S1).
Because thioethers can undergo retro-Michael reactions in
assay buffer conditions (and thus lose inhibitory potency), we
synthesized 5, which contains an all-carbon linker between the
two fragments (Chart 2). Gratifyingly, 5 was able to inhibit c-
Src with identical potency as 4. We then synthesized bivalent
inhibitors, again using an all-carbon linker, derived from the
two furan-containing fragments (EL-1061 and EL-1092) that
were identified in our enzyme-templated screen (bivalent
inhibitors 6 and 7, respectively). In addition, because both 6
and 7 are a mixture of enantiomers, we synthesized both R- and
S-enantiomers of the furan-containing amine fragments and
subsequently produced enantiopure bivalent inhibitors. The
amines were synthesized using Ellman sulfinylimine chemistry
(
2 was added at a concentration that approximates its IC20
value). After a 30 min incubation, we used a continuous activity
assay to identify fragments that had significant inhibition
difference (>2 standard deviations) between screens performed
with and without thiol 2. From the 110 acrylamide fragments,
we identified four hits that met our criteria (Figure 1, hit rate of
2
0,21
(
see Supporting Information for synthetic details).
Upon
characterization of the bivalent inhibitor series, we found that
the bivalent inhibitors with R-stereochemistry had improved
binding to c-Src (Table 1). A preference for a particular
stereochemistry indicates specific binding to c-Src, despite the
relatively weak binding observed with the acrylamide fragments.
Bivalent inhibitors 9 and 11 have 6- and 3-fold improve-
ments in binding affinity for c-Src, respectively, compared to
the control ATP-competitive fragment 3 (Table 1). We next
sought to determine whether 9 and 11 have improved
selectivity compared to fragment 3. As a measure of selectivity,
we assayed for inhibition against a small panel of homologous
kinases (c-Yes, 95% similar to c-Src; Hck, 80% similar to c-Src;
Figure 1. (left) Graph of enzyme-templated screen using c-Src and a
library of 110 acrylamides. (right) Structures of acrylamide fragment
hits.
3
.6%). Each of the four hits was confirmed in three subsequent
enzyme-templated validation screens (see Supporting Informa-
tion for details and assay controls performed).
To demonstrate that the predicted enzyme-templated
Michael additions were occurring, we performed a mass
spectrometry analysis of thiol 2 + acrylamide EL-1148 in the
presence and absence of c-Src kinase. Only when c-Src was
B
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX