A myosin-V inhibitor based on privileged chemical scaffolds

Article abstract of DOI:10.1002/anie.201004026

Switching the motor off: Privileged chemical scaffolds were used as a starting point for the development of a specific chemical inhibitor 1 of myosin-V, a key motor protein required for intracellular transport (see picture; ADP=adenosine diphosphate, ATP=adenosine triphosphate). The potency of 1, which does not compete directly with nucleotide binding, is comparable to that of other known motor-protein inhibitors used as probes in chemical biology.

Full text of DOI:10.1002/anie.201004026

Communications
DOI: 10.1002/anie.201004026
Enzyme Inhibition
A Myosin V Inhibitor Based on Privileged Chemical Scaffolds**
Kabirul Islam, Harvey F. Chin, Adrian O. Olivares, Lauren P. Saunders,
Enrique M. De La Cruz,* and Tarun M. Kapoor*
Small molecules that perturb the function of their targets on
fast time scales can be powerful probes of dynamic cellular
processes, such as intracellular transport.[1] A number of
inhibitors for motor proteins, ATPases that drive the move-
ment of cellular cargo, have been reported.[2] These chemical
inhibitors (with micromolar potency) have served as valuable
tools for the dissection of complex cellular mechanisms and
have even provided an impetus for the development of
chemotherapeutics that target motor proteins.[3] However,
chemical inhibitors are available for only approximately 6%
of the motor proteins (there are over 100 in humans[4])
involved in a variety of biological processes, including
development, hearing, intracellular signaling, and muscle
function.
Figure 1. Illustration of myosin V walking on actin filaments and
structures of compounds based on “privileged” scaffolds (bold) as
potential inhibitors (X=NH or CH2; R1–3 are various aliphatic or
aromatic groups; ADP=adenosine diphosphate, ATP=adenosine tri-
phosphate).
Myosins are motor proteins that move along the actin
cytoskeleton (Figure 1). Since their initial characterization as
proteins that drive muscle contraction, 18 different classes of
myosins have been characterized, and it is now known that
myosins are involved in almost every aspect of biological
motion.[5] However, specific small-molecule probes are avail-
able only for class II myosins.[2b,c] Therefore, we have set the
development of chemical probes for members of the other
myosin classes as our long-term goal.
The analysis of myosin structures reveals that although
these enzymes bind ATP, their structures are similar to those
of GTPases.[6] Good inhibitors for GTPases have been
generally difficult to obtain. We reasoned that the high
nucleotide affinity (low nanomolar), and not the structure of
the nucleotide-binding pocket itself, is a key factor limiting
the identification of GTPase inhibitors. This factor has also
been noted in the context of inhibitor development for
kinases that have unusually high ATP affinity.[7] Myosins have
a KM value for ATP that is typically in the micromolar range,[8]
which raises the possibility that inhibitors for these enzymes
may be more readily accessible.
To test this hypothesis, we focused on class V myosins.
These motor proteins are essential for survival in eukaryotes,
and mutations that impair activity give rise to pigmentation
and neurological defects in mice and humans.[9] The micro-
mechanics and mechanochemistry of myosin V have been the
focus of intense research.[10] However, the precise cellular
functions of myosin V remain poorly characterized, partic-
ularly in vertebrates, and a small-molecule inhibitor would be
a valuable tool.
We have shown that small molecules based on “priv-
ileged” chemical scaffolds, which map to the region of
chemical space occupied by known bioactive compounds,
can yield diverse cellular phenotypes.[11] These results, along
with other studies,[12] suggest that privileged-scaffold-based
compounds may provide efficient starting points for the
development of inhibitors of different target proteins. We
noted that such scaffolds include pyrimidines 1, oxindoles 2,
pyrrolopyrimidines 3, and pyrazolopyrimidines 4 (Figure 1).
We also noted that such scaffolds are common to many known
kinase inhibitors.[13] The specificity of kinase inhibitors is
typically examined in vitro against a large panel of known
kinases. However, the ability of these inhibitors to target
motor proteins has not been examined systematically. To
determine whether kinase inhibitors could inhibit myosin V,
[*] Dr. K. Islam, Prof. Dr. T. M. Kapoor
Laboratory of Chemistry and Cell Biology, Rockefeller University
1230 York Avenue, New York, NY 10065 (USA)
E-mail: kapoor@mail.rockefeller.edu
H. F. Chin, A. O. Olivares,[+] L. P. Saunders,
Prof. Dr. E. M. De La Cruz
Department of Molecular Biophysics and Biochemistry
Yale University, 260 Whitney Avenue, New Haven, CT 06520 (USA)
E-mail: enrique.delacruz@yale.edu
[+] Current address:
Department of Biology, Massachusetts Institute of Technology
77 Massachusetts Avenue, Cambridge, MA 02139 (USA)
[**] This research was supported by the NIH (GM71772 and GM65933
to T.M.K.; GM071688 and GM071688-03S1 to E.M.D.L.C.). T.M.K. is
a Scholar of the Leukemia and Lymphoma Society. H.F.C. was
supported by an NIH predoctoral fellowship (F31 DC009143) and,
in part, by a grant from the Yale Institute for Quantum Engineering
(awarded to E.M.D.L.C.). E.M.D.L.C. is an American Heart Associ-
ation Established Investigator (0940075N) and an NSF CAREER
Award recipient (MCB-0546353). We thank Dr. Benjamin H. Kwok
and Dr. Alexander Kelly for providing us with recombinant PLK1 and
Aurora kinase.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201004026.
8484
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2010, 49, 8484 –8488

Angewandte
Chemie
we assembled a small collection of known kinase inhibitors,
whereby we focused on various “privileged” scaffolds
(Figure 1).
effects, whereby a biaryl moiety was most favorable (see the
Supporting Information for structure–activity-relationship
(SAR) data). Further modification of the biaryl moiety led
to 7, which is the most potent compound in this series (KI
ꢀ 14 mm; Figure 3). In the pyrazolopyrimidine series, substi-
Myosin V exists as a multiprotein complex of over 12
polypeptides. It possesses two catalytic ATPase motor
domains, called heads, which bind actin filaments and
generate force (Figure 1). To test our compounds of interest,
we used a recombinant protein comprising a single ATPase
motor domain of chicken myosin Va. This construct, from a
representative member of the myosin V class, has been
extensively characterized in vitro.[14] Maximum activation of
myosin ATPase requires actin polymer. By using an enzyme-
coupled steady-state ATPase assay,[15] we found that two
compounds, 5 and 6, of 10 tested, reduced actin-activated
enzyme activity by about 35–40% at a concentration of
100 mm (Figure 2).[16] Compound 5 is a known inhibitor of
CHK1 kinase (IC50 = 7 nm),[13b] whereas compounds similar to
6 inhibit various cell-cycle kinases (IC50 values in the low-
micromolar range).[13d]
Figure 2. Initial hits 5 and 6 and their effects on the steady-state rate
of actin-activated ATP hydrolysis by single-headed myosin V (n=3,
uncertainty bars show the standard deviation; DMSO=dimethyl
sulfoxide).
Figure 3. Myosin V inhibitors 7, 8, and 9 and their dose-dependent
reduction of the rate of steady-state actin-activated ATP hydrolysis
(sÀ1 headÀ1) by single-headed myosin V (n=3, uncertainty bars show
the standard deviation).
Encouraged by these results, we synthesized a series of
compounds based on 5 and 6 to derive inhibitors that would
have decreased activity towards kinases but increased
potency for myosin V.[16] To create structural diversity, we
subjected commercially available oxindoles to a two-step
sequence of a Knoevenagel condensation and Suzuki cou-
pling, which could be carried out in either order.[13b] For the
tution changes at the 6-position improved potency, whereby
an aminobenzylthiobenzoic acid (ABTA) moiety was
favored. Our SAR study (see the Supporting Information)
indicated that a meta–meta substitution pattern of the ABTA
moiety provides the highest potency. With this position fixed,
changes at the 3-position further improved potency and led to
compound 8 as the most potent myosin V inhibitor in both
series (KI ꢀ 6 mm; Figure 3). To the best of our knowledge,
such extended aromatic substitution on oxindole and pyr-
azolopyrimidine ring systems has not been reported previ-
ously. Importantly, we could show that compound 8 also
inhibits double-headed myosin V, an active construct that
contains two catalytic domains and 12 associated light
synthesis of pyrazolopyrimidine analogues,
a four-step
sequence involving a metal–halogen exchange reaction fol-
lowed by hydrazine-mediated ring closure and nucleophilic
aromatic substitution was adopted.[13d] A total of 60 com-
pounds (39 oxindoles and 21 pyrazolopyrimidines) were
obtained with greater than 95% purity and characterized.[16]
Testing of the oxindole-based compounds indicated that
whereas substitutions at the 6-position had a modest effect on
inhibitor potency, changes at the 3-position had significant
Angew. Chem. Int. Ed. 2010, 49, 8484 –8488
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
8485

Communications
chains,[17] with equal efficiency to that of a single-headed
buffer). At a 100 mm concentration, myoVin-1 did not
significantly inhibit (< 5–10%) many representative kinases,
including CHK1, PLK1, Abl kinase, p42 MAP kinase, casein
kinase II, and Aurora kinase (Figure 4a–g). Known inhibitors
of these kinases efficiently suppressed kinase activity in our
assay (Figure 4a–g).[19]
We next examined the specificity of myoVin-1 towards
other myosins. As representative examples in other classes,
we tested skeletal-muscle myosin II and a nonmuscle myosin,
myosin VI. In in vitro ATPase assays we did not observe any
measurable inhibition by myoVin-1 at a concentration of
50 mm (Figure 5a,b), whereas blebbistatin, a known small-
motor (KI = (5.5 Æ 2.4) mm).[16]
It has been noted that protein aggregation is a common
mechanism of promiscuous chemical inhibitors.[18] Detergents
(e.g. 0.02–0.1% Triton X) have been proposed to decrease
aggregation and can be used in assays to exclude inhibitors
that act through such mechanisms. The presence of 0.1%
Triton X in the assay buffer led to an approximately 10-fold
increase in the KI value for compound 7, a result consistent
with the aggregation-based inhibition of myosin V.[16] In
contrast, compound 8 (named myoVin-1, for myosin V
inhibitor 1) showed an approximately 1.25-fold increase in
the KI value in the presence of 0.1% Triton X.[16] This
sensitivity to detergent is not likely to be significant for a
hydrophobic compound and potentially arises from the
increase in the ATPase activity of myosin V in the presence
of detergent (data not shown).
In the course of our SAR studies, we also identified
compounds that are structurally similar to myoVin-1 but
significantly less potent inhibitors of myosin V.[16] One such
compound, 9 (Figure 3), is a positional isomer of myoVin-1
and is approximately 12 times less active (KI = 72 Æ 8 mm).
These results indicate that the observed inhibition of myo-
sin V by pyrazolopyrimidine-based compounds depends on
specific features of the target protein, and are not merely a
consequence of changes in the physical properties of the
compound (e.g. solubility).
We next examined the specificity of myoVin-1. The
known SAR data for kinase inhibition by pyrazolopyrimi-
dine-based compounds suggests that myoVin-1 should have
limited activity against kinases.[13d] We tested its activity
directly by using CDK1/cyclin B, a likely target for this
chemical scaffold.[13d] In vitro kinase assays revealed that
myoVin-1 inhibited CDK1/cyclin B with at least 30-fold lower
potency (IC50 > 200 mm; Figure 4a; precise measurement was
limited by the low solubility of the compound in the assay
Figure 5. Analysis of the specificity of myoVin-1 (8) against the ATPase
activities of: a) myosin II, b) myosin VI (n=3, uncertainty bars show
the standard deviation; Blebbis.=blebbistatin).
molecule inhibitor of class II myosins,[2c] significantly inhib-
ited muscle myosin II (Figure 5a). This specificity of myoVin-
1 towards myosin V is quite remarkable given that ATPase
domains in members of the myosin superfamily are well-
conserved.[5a]
To identify the mechanism of inhibition by myoVin-1 and
identify which ATPase cycle transition(s) are targeted, we
performed a series of steady-state and transient kinetic
experiments. MyoVin-1 lowers the maximum turnover rate
Figure 4. Analysis of the specificity of myoVin-1 (8) against: a) CDK1, b) CHK1, c) PLK1, d) Abl kinase, e) p42 MAP kinase, f) casein kinase II, and
g) Aurora kinase (n=3, uncertainty bars show the standard deviation; DAP-81=Diaminopyrimidine-81,[11] Stauros.=staurosporine,[19a,b] CKII
In.=casein kinase II inhibitor,[19c] Hesp.=hesperadin[19d]).
8486 www.angewandte.org
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2010, 49, 8484 –8488

Angewandte
Chemie
(kcat) and apparent Michealis constant (KM) for actin (i.e.
uncompetitive inhibition is observed; Figure 6a). This effect
can be explained by a decrease in rate-limiting ADP release
exchanges slowly on the timescale of ADP release (i.e.
myoVin-1 binding cannot be treated as a rapid equilibrium).
A myoVin-1 affinity of (24 Æ 4) mm was obtained from the
best fit of the dependence of the amplitudes on the concen-
tration of myoVin-1. The slightly weaker KI value measured
with this assay in comparison to that found by steady-state
kinetics is probably due to the use of a modified nucleotide
analogue.[21]
These experiments indicate that myoVin-1 slows the
actin-activated myosin V ATPase by specifically inhibiting
ADP release from the actomyosin complex (Figure 7). Single
Figure 7. Mechanism of inhibition of myosin V. MyoVin-1 inhibits ADP
release from the actomyosin complex.
MANT-ATP turnover measurements[22] indicated that ATP
binding and the rate-limiting release of inorganic phosphate
(Pi) from myosin V in the absence of actin are unaffected by
myoVin-1 (data not shown), a result consistent with myoVin-1
binding to the actomyosin complex. This mechanism for the
inhibition of myosins by a chemical inhibitor is unique. We
anticipate that myoVin-1 will be a powerful tool for the
analysis of motor-protein mechanochemistry.
In summary, from a collection of privileged chemical
scaffolds, we have developed a selective myosin V inhibitor
that does not compete directly with nucleotide binding. The
potency of myoVin-1 is comparable to that of other known
motor-protein inhibitors that are extensively used as chem-
ical-biology probes.[2a,c] Many reported examples suggest that
small changes in the chemical structure of an inhibitor can
alter its mechanism of action. In particular, very minor
modifications to the chemical structure of GSK923295, an
inhibitor of the microtubule-based motor protein CENP-E,
which, like myosin, has a GTPase-like fold, change its
mechanism of inhibition from an ATP-uncompetitive to an
ATP-competitive mechanism.[23] Therefore, it is possible that
the initially tested pyrazolopyrimidines may be ATP-com-
petitive, as they can be for kinases, and that the SAR-guided
changes we introduced to obtain myoVin-1 altered the
binding mode and inhibitory mechanism. Further structural
studies will be needed to determine whether myoVin-1 binds
near the nucleotide-binding pocket or at a remote site. We
expect that the strategy we used to identify myoVin-1 should
not only be applicable to other members of the myosin
superfamily, but may be an attractive entry point into the
inhibitor-discovery cycle and thus complement the high-
throughput screening of large compound libraries. This
Figure 6. Steady-state and transient kinetic analysis. a) Effect of
myoVin-1 (8; 12 mm) on the steady-state rate of ATP hydrolysis by
myosin V at various concentrations of actin (n=3, uncertainty bars
show the standard deviation). b) Effect of myoVin-1 (8) on the
amplitude of MANT-ADP release. Data points represent the normal-
ized amplitudes of MANT-ADP release from actomyosin V–ADP. The
solid line is the best fit to a rectangular hyperbola; uncertainty bars
show the standard errors of best fits of the dissociation time course.
In the absence of myoVin-1, a residual slow phase was observed as
reported (Ampfast is the amplitude of the fast phase).[21]
from actomyosin, that is, the complex of actin and myo-
sin V.[15,20] This hypothesis was confirmed by measuring ADP
release with a fluorescent methylanthraniloyl nucleotide
analogue (MANT-ATP/MANT-ADP).[21] ADP release from
actomyosin–ADP prepared by equilibrating actomyosin and
ADP was unaffected by myoVin-1 (data not shown). How-
ever, ADP release was inhibited by myoVin-1 if actomyo-
sin V–ADP was prepared by mixing with ATP and allowing
hydrolysis to occur. This result indicates that myoVin-1 binds
to an actomyosin V intermediate populated during ATPase
cycling. When ADP release was measured in this way,
myoVin-1 lowered the amplitude, but not the rate constant
of ADP release (Figure 6b), which indicates that myoVin-1
Angew. Chem. Int. Ed. 2010, 49, 8484 –8488
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org 8487

Communications
[10] a) R. D. Vale, J. Cell Biol. 2003, 163, 445 – 450; b) J. R. Sellers, C.
Veigel, Curr. Opin. Cell Biol. 2006, 18, 68 – 73.
[11] U. Peters, J. Cherian, J. H. Kim, B. H. Kwok, T. M. Kapoor, Nat.
Chem. Biol. 2006, 2, 618 – 626.
[12] a) D. A. Horton, G. T. Bourne, M. L. Smythe, Chem. Rev. 2003,
103, 891 – 930; b) G. Mꢀller, Drug Discovery Today 2003, 8, 681 –
691; c) R. S. Bon, H. Waldmann, Acc. Chem. Res. 2010, 43, 1103 –
1114.
[13] a) G. A. Breault, R. P. A. Ellston, S. Green, S. R. James, P. J.
Jewsbury, C. J. Midgley, R. A. Pauptit, C. A. Minshull, J. A.
Tucker, J. E. Pease, Bioorg. Med. Chem. Lett. 2003, 13, 2961 –
2966; b) L. Nan-Horng, X. Ping, K. Peter, P. Chang, C. Zehan, Z.
Haiying, S. H. Rosenberg, H. L. Sham, Bioorg. Med. Chem. Lett.
2006, 16, 421 – 426; c) H.-S. Choi, Z. Wang, W. Richmond, X. He,
K. Yang, T. Jiang, T. Sim, D. Karanewsky, X.-J. Gu, V. Zhou, Y.
Liu, O. Ohmori, J. Caldwell, N. Gray, Y. He, Bioorg. Med. Chem.
Lett. 2006, 16, 2173 – 2176; d) Q. Ding, N. Jiang, J. L. Roberts,
U.S. Pat. Appl. Publ. 277655, 2005.
approach is particularly useful when the protein supply is
limited or when assays are complex and involve multiple
components (e.g. myosins), as fewer compounds need to be
tested in the primary screens. Our findings also suggest that
analysis of the specificity of kinase inhibitors should not be
limited to members of this superfamily, but off-target effects
resulting from the inhibition of proteins with significant
structural divergence, such as motor proteins, also need to be
systematically tested. Although examples of inhibitor design
through “scaffold hopping” have been reported previously,[24]
our study provides the first example of the use of kinase
inhibitors to develop compounds that target a protein with a
GTPase-like fold.
Received: July 1, 2010
Revised: August 5, 2010
[14] E. M. De La Cruz, A. L. Wells, S. S. Rosenfeld, E. M. Ostap,
H. L. Sweeney, Proc. Natl. Acad. Sci. USA 1999, 96, 13726 –
13731.
[15] E. M. De La Cruz, H. L. Sweeney, H. M. Ostap, Biophys. J. 2000,
79, 1524 – 1529.
Published online: September 28, 2010
Keywords: inhibitors · kinases · motor proteins ·
.
privileged scaffolds · structure–activity relationships
[16] See the Supporting Information for details.
[17] A. O. Olivares, W. Chang, M. S. Mooseker, D. D. Hackney, E. M.
De La Cruz, J. Biol. Chem. 2006, 281, 31326 – 31336.
[18] S. L. McGovern, E. Caselli, N. Grigorieff, B. K. Shoichet, J. Med.
Chem. 2002, 45, 1712 – 1722.
[19] a) D. M. Gadbois, J. R. Hamaguchi, R. A. Swank, E. M. Brad-
bury, Biochem. Biophys. Res. Commun. 1992, 184, 80 – 85;
b) M. W. Karaman et al., Nat. Biotechnol. 2008, 26, 127 – 132;
c) S. Sarno, H. Reddy, M. Meggio, M. Ruzzene, S. P. Davies, A.
Donella-Deana, D. Shugar, L. A. Pinna, FEBS Lett. 2001, 496,
44 – 48; d) S. Hauf, R. W. Cole, S. La Terra, C. Zimmer, G.
Schnapp, R. Walter, A. Heckel, J. van Meel, C. L. Rieder, J.-M.
Peters, J. Cell Biol. 2003, 161, 281 – 294.
[20] E. M. De La Cruz, A. L. Wells, H. L. Sweeney, E. M. Ostap,
Biochemistry 2000, 39, 14196 – 14202.
[21] D. E. Hannemann, W. Cao, A. O. Olivares, J. P. Robblee, E. M.
De La Cruz, Biochemistry 2005, 44, 8826 – 8840.
[22] E. M. De La Cruz, H. M. Ostap, Methods Enzymol. 2009, 455,
157 – 192.
[23] K. W. Wood et al., Proc. Natl. Acad. Sci. USA 2010, 107, 5839 –
5844.
[1] a) J. R. Peterson, T. J. Mitchison, Chem. Biol. 2002, 9, 1275 –
1285; b) S. L. Schreiber, Chem. Eng. News 2003, 81, 51 – 61.
[2] a) T. U. Mayer, T. M. Kapoor, S. J. Haggarty, R. W. King, S. L.
Schreiber, T. J. Mitchison, Science 1999, 286, 971 – 974; b) A.
Cheung, J. A. Dantzig, S. Hollingworth, S. M. Baylor, Y. E.
Goldman, T. J. Mitchison, A. F. Straight, Nat. Cell Biol. 2002, 4,
83 – 88; c) A. F. Straight, A. Cheung, J. Limouze, I. Chen, N. J.
Westwood, J. R. Sellers, T. J. Mitchison, Science 2003, 299, 1743 –
1747; d) G. Bergnes, K. Brejc, L. Belmont, Curr. Top. Med.
Chem. 2005, 5, 127 – 145.
[3] M. A. Lampson, T. M. Kapoor, Nat. Chem. Biol. 2006, 2, 19 – 27.
[4] R. D. Vale, Cell 2003, 112, 467 – 480.
[5] a) J. A. Sellers, Myosins, 2nd ed., Oxford University Press,
Oxford, 1999; b) M. Krendel, M. S. Mooseker, Physiology 2005,
20, 239 – 251.
[6] a) C. A. Smith, I. Rayment, Biophys. J. 1996, 70, 1590 – 1602;
b) R. D. Vale, J. Cell Biol. 1996, 135, 291 – 302.
[7] Z. A. Knight, K. M. Shokat, Chem. Biol. 2005, 12, 621 – 637.
[8] a) D. D. Hackney, P. K. Clark, J. Biol. Chem. 1985, 260, 5505 –
5510; b) S. Watanabe, K. Mabuchi, R. Ikebe, M. Ikebe,
Biochemistry 2006, 45, 2729 – 2738.
[24] N. Winssinger, S. Barluenga, M. Karplus, WO/2008/021213, 2008.
[9] a) S. L. Reck-Peterson, D. W. Provance, Jr., M. S. Mooseker,
J. A. Mercer, Biochim. Biophys. Acta Mol. Cell Res. 2000, 1496,
36 – 51; b) K. M. Trybus, Cell. Mol. Life Sci. 2008, 65, 1378 – 1389.
8488 www.angewandte.org
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2010, 49, 8484 –8488