Examining the mechanism of action of a kinesin inhibitor using Stable Isotope Labeled Inhibitors for Cross-Linking (SILIC)

Article abstract of DOI:10.1021/ja204561q

It is difficult to determine a chemical inhibitor's binding site in multiprotein mixtures, particularly when high-resolution structural studies are not straightforward. Building upon previous research involving photo-cross-linking and the use of mixtures of stable isotopes, we report a method, Stable Isotope Labeled Inhibitors for Cross-linking (SILIC), for mapping a small molecule inhibitor's binding site in its target protein. In SILIC, structure-activity relationship data is used to design inhibitor analogues that incorporate a photo-cross-linking group along with either natural or 'heavy' stable isotopes. An equimolar mixture of these inhibitor analogues is cross-linked to the target protein to yield a robust signature for identifying inhibitor-modified peptide fragments in complex mass spectrometry data. As a proof of concept, we applied this approach to an ATP-competitive inhibitor of kinesin-5, a widely conserved motor protein required for cell division and an anticancer drug target. This analysis, along with mutagenesis studies, suggests that the inhibitor binds at an allosteric site in the motor protein.

Full text of DOI:10.1021/ja204561q

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Examining the Mechanism of Action of a Kinesin Inhibitor Using
Stable Isotope Labeled Inhibitors for Cross-Linking (SILIC)
Sarah A. Wacker, Sudhir Kashyap, Xiang Li,* and Tarun M. Kapoor*
Laboratory of Chemistry and Cell Biology, The Rockefeller University, New York, New York, 10065
S Supporting Information
b
ABSTRACT: It is difficult to determine a chemical inhibi-
tor’s binding site in multiprotein mixtures, particularly when
high-resolution structural studies are not straightforward.
Building upon previous research involving photo-cross-linking
and the use of mixtures of stable isotopes, we report a
Figure 1. Schematic for SILIC. Inhibitors, with a photo-cross-linking
method, Stable Isotope Labeled Inhibitors for Cross-linking
(SILIC), for mapping a small molecule inhibitor’s binding
site in its target protein. In SILIC, structureꢀactivity
relationship data is used to design inhibitor analogues that
incorporate a photo-cross-linking group along with either
natural or ‘heavy’ stable isotopes. An equimolar mixture of
these inhibitor analogues is cross-linked to the target protein
to yield a robust signature for identifying inhibitor-modified
peptide fragments in complex mass spectrometry data. As a
proof of concept, we applied this approach to an ATP-
competitive inhibitor of kinesin-5, a widely conserved motor
protein required for cell division and an anticancer drug
target. This analysis, along with mutagenesis studies, sug-
gests that the inhibitor binds at an allosteric site in the motor
protein.
substituent and natural (N) or heavy (H) isotopes, are mixed with a
complex of proteins, including the target (green). After UV-cross-
linking, protein digestion, and LCꢀMS one obtains complex mass spectra
that can be filtered based upon a ‘signature’ of peaks with the expected
mass difference and equal signal intensities.
In many cases, however, the inhibitor’s dual modifications, for
photo-cross-linking and affinity-capture, can alter the compound’s
mechanism of action. As an alternative approach to identify in-
hibitorꢀprotein adducts within complex mass spectra, inhibitor
analogues can be generated such that they carry a unique isotope
pattern.11,12 The incorporation of natural and ‘heavy’ stable
isotopes into a benzophenone photo-cross-linker moiety ap-
pended to the inhibitor of interest has been shown to aid the
identification of its target in a proof of concept study.13 However,
the method is not likely to be useful for mapping an inhibitor’s
binding site. This is, in large part, due to the cross-linking group
being incorporated via a linker, so that it is a significant distance
from the functional groups that are likely to make key contacts
with the target’s binding site.
Here, building on these studies, we have developed a method,
named stable isotope labeled inhibitors for cross-linking (SILIC),
for mapping small-moleculeꢀprotein binding sites. In the first
step of this approach we incorporate a photo-cross-linking group
(e.g., azide) into the inhibitor of interest (Figure 1), guided by
available structureꢀactivity relationship (SAR) data. The photo-
cross-linking group is appended at a site that does not change the
inhibitor’s mechanism of action, but is in the closest proximity
possible to the inhibitor’s activity-conferring functionality, so as
to increase the probability that cross-links are at, or near, the
protein’s inhibitor-binding pocket. Natural and heavy isotope
inhibitor analogues, which have a mass difference of a few daltons
but otherwise identical physical properties, are then generated.
The multiprotein complex to be analyzed is then incubated with a
1:1 mixture of natural and heavy inhibitor. After photo-cross-
linking and protein digestion, the resulting mixture of peptide
fragments is separated by HPLC and analyzed using high-
resolution mass spectrometry. The resulting mass spectra can
comprise thousands of peaks, and the peptideꢀinhibitor adduct
ioactive small molecules can be important chemotherapeutic
B
drugs as well as valuable tools to elucidate the cellular func-
tions of their target proteins.1,2 In both contexts, the value of the
small molecule can be limited by a lack of understanding of its
mechanism of inhibition and mode of target protein binding.
Without these data, it can be difficult to improve potency,
evaluate specificity, and fully explain cellular phenotypes result-
ing from drug treatment. A precise understanding of how a
bioactive molecule interacts with its target can address these
issues,3 but in many cases a crystal of the drug bound to the
protein is difficult to obtain. Moreover, when the small molecule’s
target is part of a multiprotein complex, analyzing the mechanism
of inhibition using structural approaches can be challenging.
Photo-cross-linking of small molecules to proteins has been
used to trap drugꢀtarget protein interactions in complex protein
mixtures.4ꢀ6 Identifying drug targets and mapping drug binding
sites after photo-cross-linking typically relies on systematic mass
spectrometry-based analyses of digested protein fragments to
identify those with a small-molecule adduct.7,8 While there are
examples of the successful use of this approach, the general ap-
plicability of the method has been limited as cross-linking is often
substoichiometric9 and the different possible inhibitorꢀpeptide
adducts can be difficult to detect in complex mass spectra. One
strategy to address this involves generating inhibitor analogues
with an affinity tag for capturing the inhibitorꢀpeptide adducts.10
Received: May 18, 2011
Published: July 15, 2011
r
2011 American Chemical Society
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the inhibitorꢀkinesin interaction in this context provided an in-
teresting case for developing and applying our approach.
Guided by available SAR data,14 we designed analogues of 1,
which contain an azide substituent on the phenyl ring as a photo-
reactive group and have natural (hydrogen, H; compound 2) and
heavy (deuterium, D; compound 3) isotopes (Figure 2a). Synth-
esis of compound 2 was based on the published procedure for
1.14 To incorporate deuterium atoms that generate a 4 Da mass
difference compared with 2, toluene-d8 was used as a starting
material to synthesize the substituted phenyl ring moiety in 3
(see Supporting Information). Notably, the introduction of the
cross-linking group in 1 did not affect the potency of the com-
pounds and, as expected, the natural and heavy analogues have
similar activities against kinesin-5 (Figure 2b, IC50:2=1.2(0.4 μM;
3 = 1.0 ( 0.4 μM). A 1:1 mixture of 2 and 3 (3 μM each) was
incubated with kinesin-5 ATPase domain and microtubules.
After UV irradiation at 254 nm for 30 min, the reaction mixture
was resolved by SDS-PAGE. Following in-gel digestion, the peptide
mixture was separated and analyzed by LCꢀMS/MS. Computer-
based analysis (MaxQuant26) was used to efficiently detect peaks
with a mass difference of 4 Da that eluted at the same time from
the LC column. In independent experiments (n = 3), we found
equal intensity peaks corresponding to a single peptide (Figure 3a),
indicating that this was likely to be the major inhibitorꢀpeptide
adduct. Further analysis by MS/MS identified this peptide as a
fragment corresponding to Ser120ꢀArg138 of kinesin-5 and the
cross-linking site of the inhibitors is very likely at one of three
amino acid residues (Tyr125, Thr126, or Trp127) (Figure 3b).
We next examined whether excess 1 can suppress the cross-
linking of 2 (or 3) to kinesin-5. For this experiment, we cross-linked
kinesin-5 with 2 (3 μM) in the presence of excess 1 (45 μM) and,
in parallel, cross-linked kinesin-5 with 3 (3 μM) alone. These
samples were then mixed and processed for mass spectrometry
analysis. This protocol provided a quantitative readout of the
competition, as only one set of peaks in the mass spectrum should
be suppressed. As shown in Figure 3c, 1 competes with 2, sug-
gesting that 1 also binds at a site proximal to residues Tyr125,
Thr126, and Trp127.
In the three-dimensional structure of the kinesin-5 ATPase
domain (Figure 4a, PDB: 2WOG27), residues Y125, T126, and
W127 map to a portion of loop-5. This loop is part of the
allosteric binding site of other kinesin-5 inhibitors,21,27 such as
S-Trityl-L-cyteine (STLC, IC50 = 1 μM, steady-state ATPase
assay without microtubules28). As these inhibitors bind this
pocket in the absence of microtubules, we examined if 1 did
the same. To test this, we repeated the cross-linking and competition
experiments in the absence of microtubules and found the same
single cross-linking site (Figure S1). Furthermore, we found that ad-
dition of excess STLC (50 μM) prevented cross-linking of 2(5μM)
to kinesin-5, relative to the reference sample (kinesin-5 cross-
linked to 3, Figure 4b). Together, these data suggest that 1, like
STLC, binds in an allosteric site in kinesin-5.
We next used site-directed mutagenesis to analyze whether
inhibition of kinesin-5 by 1 was sensitive to changes in the allosteric
pocket. A leucine-214 to alanine (L214A) mutation, in this allosteric
binding site of kinesin-5, is known to suppress STLC inhibition
(Figure 4a).29 Using the steady-state ATP hydrolysis assay, both
with and without microtubules, we found that this mutation led
to a 10-fold increase in the potency of 1 (Figure 4c, d, Table S1).
Importantly, cross-linking of 2 and 3 to kinesin-5, which had the
L214A mutation, resulted in identification of the same binding site
(Figure S2). In addition, we found that 1 is an ATP-competitive
Figure 2. (a) Chemical structure of an ATP-competitive kinesin-5
inhibitor and analogues generated for SILIC. (b) Potency of compounds
1, 2, and 3 in inhibiting kinesin-5 activity, as examined using a steady-
state microtubule-stimulated ATP hydrolysis assay. (c) In the presence
of MgATP (1 mM) and DMSO, homotetrameric kinesin-5 drives
microtubule gliding at an average rate of 19.6 ( 4.5 nm/s (left panel,
n > 25). 1 (50 nM) inhibits this activity (right panel, n > 30). Scale
bar is 2 μm.
is likely to be of low abundance due to substoichiometric labeling.
The peptideꢀinhibitor adduct is identified when a pair of
peptides that coelute in the LC have the expected mass difference
and essentially equal signal intensity. Finally, guided by these data,
site-directed mutagenesis experiments can be designed to further
examine the inhibitor-binding sites identified by SILIC.
As a proof of concept, we focused on compound 1, an inhibitor
of kinesin-5 (Figure 2a).14 Kinesins, which comprise a family of
over 40 proteins, are motor proteins that move cargo along
microtubules, polymers of the cytoskeletal protein tubulin.15,16
The kinesin-5 family is required for the assembly of the micro-
tubule-based apparatus necessary for cell division.17 Inhibitors of
kinesin-5 have provided valuable insight into mechanisms of cell
division and have entered clinical trials as anticancer drugs.18,19
Kinesin-5 inhibitors that are in clinical trials, and have been used
for cytological experiments, bind an allosteric site not conserved
in other kinesins.20,21 These inhibitors are not competitive with
respect to ATP.22 Recently, ATP-competitive inhibitors of
kinesin-5 have been reported, including compound 1.14,23 As
the ATP-binding site is the most conserved feature in kinesins,
the possibility arises that these inhibitors may provide valuable
starting points for developing new inhibitors for other kinesins.
However, the binding site of 1 in kinesin-5 is not known, and
structural data have been difficult to obtain.14
To map the binding site of 1, we first analyzed its mechanism
of action. We find that 1 inhibits steady state ATP hydrolysis by
human kinesin-50s ATPase domain (residues 1ꢀ368, expressed
in bacteria) 25-times more potently when microtubules, the
motor protein’s tracks, are present in the reaction (IC50: 1 + micro-
tubules + kinesin-5 = 1.2 ( 0.3 μM; 1 + kinesin-5 = 30 ( 7 μM;
Figure 2b, Table S1). We next examined inhibition of kinesin-5
driven microtubule gliding. These assays require protein con-
structs that are larger than those consisting of the monomeric
ATPase domain. We generated full-length homotetrameric ki-
nesin-5 (Xenopus laevis, expressed in insect cells as published
previously24) and found that it drives microtubule gliding at
19.6 ( 4.5 nm/s (1 mM MgATP). Remarkably, even at 50 nM of
1, the motor activity was completely inhibited (Figure 2c),
suggesting that tightly bound motor proteinꢀmicrotubule com-
plexes are formed in the presence of 1 and these complexes act as
‘brakes’ against other active motor protein molecules to stop
microtubule motion.25 Together, these ATPase and microtubule
gliding assay data suggest that the binding mode of 1 to kinesin-5
should be examined in the presence of microtubules. Analyzing
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Figure 4. (a) Kinesin-5 ATPase domain (green, PDB: 2WOG27) in
complex with ADP (orange) and STLC (blue). The cross-linked re-
sidues Y125, T126, and W127 are shown in red, while residue L214 is in
purple. (b) Cross-linking of 2 (5 μM) to kinesin-5 in the presence of
STLC (50 μM). (c) Representative curve of dose-dependent inhibition
of steady-state microtubule-stimulated ATP hydrolysis by wildtype (wt)
kinesin-5 and the L214A mutant protein. (d) Representative curve of
dose-dependent inhibition of steady-state ATP hydrolysis by wildtype
(wt) kinesin-5 and the L214A mutant protein, in the absence of micro-
tubules. IC50 values are averages ( s.d. (n = 3).
Figure 3. (a) A representative mass spectrum of the ‘signature’ peaks
that denote peptide fragments of kinesin-5 cross-linked by equimolar
2 (pink circles) and 3 (blue squares) in the presence of microtubules.
(b) The MS/MS spectrum and summary of fragmented ions of the
cross-linked peptide. (c) When 2 (3 μM) is cross-linked in the presence
of 1 (45 μM), there is near complete loss of cross-linked peptide, relative
to that observed in the presence of 3 (3 μM) alone. Cartoon shows
kinesin-5 (green) and microtubules (red) with cross-linking compounds.
feasible. However, aryl groups appear at high frequency in bioactive
small molecules. In these cases, SAR studies could allow the in-
clusion of an azide moiety such that it does not alter the inhibitor’s
mechanism of action and yet is proximal to the target’s binding
site residues. In the future we wish to extend this analysis to more
complex cellular contexts to examine whether cellular physiology
alters the mode of drugꢀtarget interaction and mechanism of
drug action.
inhibitor of the mutant kinesin-5 (L214A) (Figure S3), indicat-
ing that the mode of inhibition is not altered by this mutation.
Together, our data are consistent with 1 binding at, or close to,
the site bound by other kinesin-5 inhibitors that are not ATP-
competitive.
It is relatively uncommon for nucleotide-competitive inhibi-
tors to bind at an allosteric site in an ATPase.30 Interestingly,
another example of this mechanism of inhibition has been described
for another kinesin-5 inhibitor, GSK-1.23 This compound has
been proposed to bind to an allosteric site that is distinct from the
binding site of compound 1 and STLC. Further studies with both
kinesin-5 ATP-competitive inhibitors will be needed to reveal
how binding at allosteric sites results in ATP-competitive inhibi-
tion that leads to a tightly bound microtubuleꢀmotor protein
complex.
’ ASSOCIATED CONTENT
S
Supporting Information. Details of the synthesis of com-
b
pounds 2 and 3, experimental findings from cross-linking without
microtubules and with the L214A mutant, ATP-competition
results, experimental procedures, and complete refs 5,8,23. This
material is available free of charge via the Internet at http://pubs.
acs.org.
’ AUTHOR INFORMATION
Corresponding Author
xiangli@hku.hk; kapoor@rockefeller.edu
In summary, we demonstrate that SILIC can be used to map
the binding site of an inhibitor to its target. We believe this approach
should be effective in analyzing inhibitorꢀtarget interactions,
particularly when structural studies are intractable. A potential
limitation of the approach is that the incorporation of a photo-
cross-linking moiety, such as an aryl azide, may not always be
’ ACKNOWLEDGMENT
We thank Brian Chait, Kelly Molloy, and Yinyin Li for reagents,
equipment use, technical assistance, and discussions. This work
was supported by the NIH (GM65933).
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