Discovery of a type III inhibitor of LIM kinase 2 that binds in a DFG-out conformation

Article abstract of DOI:10.1021/ml500242y

The first allosteric, type III inhibitor of LIM-kinase 2 (LIMK2) is reported. A series of molecules that feature both an N-phenylsulfonamide and tertiary amide were not only very potent at LIMK2 but also were extremely selective against a panel of other k

Full text of DOI:10.1021/ml500242y

Letter
pubs.acs.org/acsmedchemlett
Discovery of a Type III Inhibitor of LIM Kinase 2 That Binds in a DFG-
Out Conformation
Nicole C. Goodwin,*,†,§ Giovanni Cianchetta,† Hugh A. Burgoon,† Jason Healy,† Ross Mabon,†
Eric D. Strobel,† Jason Allen,‡ Shuli Wang,‡ Brian D. Hamman,‡ and David B. Rawlins*,†
†Lexicon Pharmaceuticals, 350 Carter Road, Princeton, New Jersey 08540, United States
‡Lexicon Pharmaceuticals, 8800 Technology Forest Place, The Woodlands, Texas 77381, United States
S
* Supporting Information
ABSTRACT: The first allosteric, type III inhibitor of LIM-kinase 2 (LIMK2) is
reported. A series of molecules that feature both an N-phenylsulfonamide and tertiary
amide were not only very potent at LIMK2 but also were extremely selective against a
panel of other kinases. Enzymatic kinetic studies showed these molecules to be
noncompetitive with ATP, suggesting allosteric inhibition. X-ray crystallography
confirmed that these sulfonamides are a rare example of a type III kinase inhibitor that
binds away from the highly conserved hinge region and instead resides in the
hydrophobic pocket formed in the DFG-out conformation of the kinase, thus
accounting for the high level of selectivity observed.
KEYWORDS: LIM kinase 2, DFG-out, type III kinase inhibitor, allosteric, selective, noncompetitive
rotein kinases continue to be of interest in the
Ppharmaceutical industry because of their roles in
phosphorylation cascades, which control many cellular
processes within the cell. Deregulation of protein kinase
activity has been implicated in a variety of therapeutic areas,
including cancer, metabolic disorders, and immunology.1
Inhibitors of kinases have been pursued for many years
resulting in a number of approved small molecule drugs.2 As a
better understanding of kinase inhibitors has been developed,
five different classes of inhibitors have been identified, in
addition to covalent inhibitors. Type I inhibitors bind in the
ATP-binding pocket and compete with the adenine moiety of
ATP for the essential hydrogen bond interactions in the “hinge
region”. Type II inhibitors were defined with the discovery of
imatinib.3 These inhibitors not only occupy the ATP-binding
pocket but also extend into a hydrophobic, allosteric pocket
that is formed when the Asp-Phe-Gly (DFG) residues of the
activation loop fold out (“DFG-out”) from their normal
position and stabilize an inactive conformation of the kinase.4−7
Types III−V inhibitors have been defined more recently. Type
III inhibitors bind in the catalytic domain of the kinase near the
ATP-binding pocket but do not interact with the hinge region
and include molecules that bind in the hydrophobic pocket
formed in the DFG-out confirmation.7−10 Type 4 inhibitors
bind in a truly allosteric site away from the catalytic domain of
the kinase.11,12 Lastly, type V inhibitors are bivalent or
bisubstrate compounds.13,14 The residues in the catalytic
domain of kinases are highly conserved, especially in the
ATP-binding pocket, making selectivity particularly difficult to
achieve with type I and some type II inhibitors. Thus,
identification of allosteric inhibitors that do not occupy the
hinge region of the ATP-binding pocket would provide
molecules with a greater level of specificity for their kinase
target and minimize off-target pharmacology.
LIM-kinases (LIMK) 1 and 2 have been implicated as key
regulators of the dynamics of actin polymerization in the
cytoskeleton due to their role in the phosphorylation and
subsequent deactivation of cofilin, a protein that depolymerizes
the actin filaments.15 LIM-kinases have been investigated for
their role in a variety of therapeutic indications, including
cancer16 and, as we previously reported, open-angle glauco-
ma.17 In an effort to identify an alternative chemical series to
inhibit LIMK2, high-throughput screening identified sulfona-
mide 1 as a low micromolar inhibitor. The low molecular
weight and modular structure that would be amenable to a
rapid structure−activity relationship (SAR) made it an
attractive candidate for further investigation.
Our initial lead optimization efforts focused on the
sulfonamide side. Reversal of the S-thiophenylsulfonamide
linkage of 1 to the N-phenylsulfamoyl linker of 2 resulted in an
800-fold increase in potency (Figure 1). With this low
nanomolar inhibitor in hand, further in vitro characterization
was done to establish its selectivity profile against a panel of
representative kinases from the seven kinase families at low
millimolar physiological ATP concentration,18,19 while the
LIMK2 biochemical assay was run at a slightly lower
Special Issue: New Frontiers in Kinases
Received: June 10, 2014
Accepted: August 7, 2014
Published: August 7, 2014
© 2014 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
Menten constant (Km) is unaffected and Vmax decreases as the
concentration of sulfonamide 2 increases. In contrast, as shown
in Figure 2b, titration of the known ATP competitor of LIMK2
results in a linear increase in the ATP Km but no change in
22
Vmax
.
A Lineweaver−Burk plot (Figure 3) was completed to
Figure 1. Lead identification of arylsulfonamide series.
concentration of ATP.20 As ATP has a relatively weak affinity
for LIMK2 (Km = 85 μM) compared to other kinases, overall
selectivity of compound 2 will likely increase at the higher
physiological ATP concentrations. As shown in Chart 1,
sulfonamide 2 was surprisingly selective for LIMK2 (39 nM) in
this screen and even had almost a 100-fold level of selectivity
against the closely related LIMK1 (3.2 μM).
a
Chart 1. Selected Kinase Panel (μM)
Figure 3. Lineweaver−Burk plot of the inverse reaction rate against
the inverse concentration of ATP, where each line represents a
different concentration of sulfonamide 2.
further characterize the type of enzyme inhibition. The plots for
six different concentrations of inhibitor 2 intersect on the X-axis
but have different slopes and points of intersection on the Y-
axis. This suggests that 2 is functioning as a noncompetitive
inhibitor LIMK2, rather than an uncompetitive (lines never
intersect), competitive (lines intersect on the Y-axis), or mixed
inhibitor (lines intersect somewhere in between the X- and Y-
axes).23,24
The excitement over this chemical series of selective,
allosteric inhibitors prompted a rapid expansion of the SARs
to increase the potency at LIMK2. The synthetic strategy,
outlined in Scheme 1, was straightforward. Substituted anilines
a
LIMK2 assays conducted in the presence of 300 μM ATP. Other
assays were conducted in the presence of 1 mM ATP.
The spectacular level of selectivity of sulfonamide 2 led us to
examine the nature of the inhibition of this compound. This
level of selectivity is rare among kinase inhibitors, and thus, it
was proposed that this selectivity could be explained if
sulfonamide 2 was binding in an allosteric manner away from
the more conserved hinge region of LIMK2 and thus would not
bind competitively against ATP. To determine whether or not
sulfonamide 2 competes with ATP for binding to LIMK2,
kinetic experiments varying both ATP levels and inhibitor
concentrations were performed using 2 and a known type I
LIMK2 inhibitor.21 As shown in Figure 2a, the ATP Michaelis−
Scheme 1. General Procedure for Sulfonamides
were reacted with commercially available 4-(chlorosulfonyl)-
benzoic acid 3 to form N-aryl sulfonamides 4. These
sulfonamides were then reacted with substituted amines in
the presence of 1-ethyl-3-(3- dimethyl-aminopropyl) carbodii-
mide (EDCI) and N-hydroxybenzotriazole (HOBt) to provide
functionalized amides 5.
SAR studies began with the N-phenyl of the sulfonamide.
Replacements of the phenyl group with heteroaryl, alkyl, or
cycloalkyl groups were detrimental to potency (not shown).
Changes to the aryl group of the sulfonamide are shown in
Table 1. In general, only minor substitutions to the phenyl ring
were permitted. Addition of groups at the ortho position (6−8)
and para position (9−11) led to a decrease in LIMK2 activity.
Small groups such as fluoro (12) and methyl (14) installed at
the meta position did provide a slight improvement in activity,
Figure 2. (a) Enzyme kinetics of sulfonamide 2. (b) Enzyme kinetics
of a known ATP competitive LIMK2 inhibitor. See text for more
detailed description.
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were equipotent or 2-fold more potent, respectively, than the
parent compound 2. However, pendant basic amines, such as
piperidine 23, led to a marked decrease in potency.
Replacement of the benzyl group with a saturated piperdine
moiety retained submicromolar potency as long as the nitrogen
remained electron deficient (24 and 25), but when the amine
was rendered basic by simple N-alkyl substitution (27 and 28),
compounds lost all activity at LIMK2. Lastly, substitution at the
methylene position on the benzyl was investigated. Small
groups like methyl were tolerated (enantiomers 29 and 30), but
larger groups lost potency (data not shown).
Table 1. SAR of the Aryl Sulfonamide
a
cmpd
R1
R2
R3
R4
LIMK2 IC50 (μM)
2
H
H
F
H
H
H
H
H
H
H
F
H
H
H
H
F
0.039
0.052
0.12
1.0
6
H
7
H
CH3
OCH3
H
8
H
SAR at the N-methyl position of the benzyl amide of 2 was
investigated (Table 3). Similar to the benzyl position, the scope
9
H
0.081
3.2
10
11
12
13
14
15
16
17
18
H
H
CH3
OCH3
H
H
H
24.1
0.022
0.051
0.022
0.54
>27
Table 3. N-Substitution SAR of the Benzyl Amide
H
H
H
H
CI
H
H
H
CH3
OCH3
t-Bu
NMe2
H
H
H
H
H
H
H
H
H
H
H
>27
a
cmpd
R
LIMK2 IC50 (μM)
CH3
H
H
0.095
a
2
Me
Et
0.0039
0.003
0.019
0.052
0.16
IC50 values are averages of 4−8 indepedent IC50 measurements.
31
32
33
34
35
36
(CH2)2OH
(CH2)2CN
(CH2)2NMe2
(CH2)2NH2
(CH2)3NH2
but as the size continued to increase, potency abated (16 and
17). Capping of the amide with a methyl group (18) also
provided a less active compound.
Unlike the limited SAR exhibited in Table 1, the tertiary
amide on the left side of the molecule had a greater scope
(Table 2). While the amide itself was irreplaceable, the benzyl
group could be elaborated or replaced with a saturated ring
system. In particular, the para position on the benzyl group
could tolerate a wide variety of substitutions. Installation of
neutral groups, as shown in ether 20 and carboxylic acid 21,
1.2
1.5
a
IC50 values are averages of 4−8 indepedent IC50 measurements.
of this position allowed for a variety of elongated, neutral
moieties to be incorporated while retaining potency at LIMK2
(31−33), but basic amines were less potent (34−36). The
simple exchange of N-methyl for N-ethyl (31) added a 10-fold
increase in potency, providing the most potent compound
synthesized in this series at 3 nM.
Table 2. SAR of the Benzyl Group of the Amide
Finally, we hypothesized that a 1,2- or 1,3-disubstituted
heterocycle could be a viable isosteric replacement for the
amide. 1,2-Disubstitution patterns were found to be preferred
over 1,3-disubstitution (data not shown). Various 1,2-
disubstituted heterocycles are shown in Table 4. The most
potent isosteres kept a hydrogen acceptor that mapped onto
the oxygen of the amide carbonyl. Imidazole 39 was the most
potent of this series, while pyridine 40 and pyrazine 41 also
retained LIMK2 potency. However, none of these compounds
were more potent than the simple N-ethyl-N-benzylamide 31.
Because of its good aqueous solubility and the decent
potency, compound 22 was selected to be cocrystallized with
LIMK2. The X-ray structure was obtained and confirmed the
type III binding mode of these sulfonamide inhibitors. As
shown in Figure 4, ligand 22 is exclusively binding in the
hydrophobic pocket formed when the DFG residues of the
activation loop are in the DFG-out conformation. The carbonyl
of the amide participates in an interaction with the backbone
N−H of D469 of the DFG residues in the activation loop, thus
explaining the observation that a hydrogen bond acceptor was
critical at this position. Meanwhile, the sulfonamide carbonyl
further anchors the ligand by forming a hydrogen bond with
the R474 residue adjacent to the DFG motif on the activation
loop. The nonpolar phenyl group of the sulfonamide
contributes an additional hydrophobic interaction by occupying
the newly formed hydrophobic cleft. Occupation of this small
a
IC50 values are averages of 4−8 indepedent IC50 measurements.
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ACS Medicinal Chemistry Letters
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Table 4. Heteroaryl Isosteres for the Amide Group
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental details and characterization of compounds in this
letter, detailed information about the biological activity assays,
and crystallographic information. This material is available free
of charge via the Internet at http://pubs.acs.org.
Accession Codes
Coordinates for the LIMK2-22 structure have been deposited
in the Protein Data Bank with accession code 4TPT.
AUTHOR INFORMATION
Corresponding Authors
*(N.C.G.) E-mail: nicole.c.goodwin@gsk.com.
*(D.B.R.) E-mail: drawlins@lexpharma.com.
■
Present Address
§(N.C.G.) GlaxoSmithKline, 20 T. W. Alexander Drive,
Research Triangle Park, North Carolina 27709, United States.
Author Contributions
The manuscript was written through contributions of all
authors.
Notes
a
IC50 values are averages of 4−8 indepedent IC50 measurements.
The authors declare no competing financial interest.
ABBREVIATIONS
■
LIMK2, LIM Kinase 2; ATP, adenosine triphosphate; Km,
Michaelis−Menten constant; Vmax, maximum rate achieved at
saturating substrate concentration; SAR, structure−activity
relationship; IC50, half maximal inhibitory concentration;
THF, tetrahydrofuran; EDCI, 1-ethyl-3-(3-dimethyl-amino-
propyl) carbodiimide; HOBt, N-hydroxybenzotriazole
REFERENCES
■
(1) Cohen, P. Protein kinases: the major drug targets of the twenty-
first century? Drug Discovery 2002, 1, 309−315.
(2) Zhang, J.; Yang, P. L.; Gray, N. S. Targeting cancer with small
molecule kinase inhibitors. Nature 2009, 9, 28−37.
(3) Schindler, T.; Bornmann, W.; Pellicena, P.; Miller, W. T.;
Clarkson, B.; Kuriyan, J. Structural mechanism for STI-571 inhibition
of abelson tyrosine kinase. Science 2000, 289, 1938−1942.
(4) Zhao, Z.; Wu, H.; Wang, L.; Liu, Y.; Knapp, S.; Liu, Q.; Gray, N.
S. Exploration of type II binding mode: A privileged approach for
kinase inhibitor focused drug discovery. ACS Chem. Biol. 2014, 9,
1230−1241.
(5) Blanc, J.; Geney, R.; Menet, C. Type II kinase inhibitors: An
opportunity in cancer for rational design. Anti-Cancer Agents Med.
Chem. 2013, 13, 731−747.
(6) Okaniwa, M.; Hirose, M.; Imada, T.; Ohashi, T.; Hayashi, Y.;
Miyazaki, T.; Arita, T.; et al. Design and synthesis of novel DFG-out
RAF/vascular endothelial growth factor receptor 2 (VEGFR2)
inhibitors: Exploration of [5,6]-fused bicyclic scaffolds. J. Med. Chem.
2012, 55, 3452−2478.
(7) Richters, A.; Nguyen, H. D.; Phan, T.; Simard, J. R.; Gruetter, C.;
Engel, J.; Ruah, D. Identification of type II and III DDR2 inhibitors. J.
Med. Chem. 2014, 57, 4252−4262.
(8) Fasano, M.; Della Corte, C. M.; Califano, R.; Capuano, A.;
Troilani, T.; Marinelli, E.; Ciardiello, F.; Morgillo, F. Type III or
allosteric kinase inhibitors for the treatment of non-small cell lung
cancer. Exp. Opin. Invest. Drugs. 2014, 23, 809−821.
(9) Garuti, L.; Roberti, M.; Bottegoni, G. Non-ATP competitive
protein kinase inhibitors. Curr. Med. Chem. 2010, 17, 2804−2821.
(10) Dietrich, J.; Hulme, C.; Hurley, L. H. The design, synthesis, and
evaluation of eight hybrid DFG-out allosteric kinase inhibitors: A
structural analysis of the binding interactions of Gleevec, Nexavar, and
BIRB-796. Biorg. Med. Chem. 2010, 18, 5738−5748.
Figure 4. X-ray crystal structure with ligand 22. The ligand is shaded
gray, while the DFG residues are shaded yellow. Key hydrogen bonds
are represented as dotted gray lines.
hydrophobic pocket can account for the stringent SAR
observed at this position (see Table 1). Meanwhile, the N-
benzylamide moiety is pointed out to the solvent front and thus
explains the diversity that was tolerated at this position (see
Table 2). As can be seen, the inhibitor 22 does not extend past
the gatekeeper residue T316 and therefore has no interaction
with the hinge region, which is located in the area to the left of
the structure depicted in Figure 4.
In summary, we have discovered and characterized a novel
series of sulfonamides, which are potent and extremely selective
inhibitors of LIMK2. A series of kinetic experiments showed
these inhibitors to be non-ATP competitive. X-ray crystallog-
raphy further demonstrated that these molecules were binding
as type III kinase inhibitors that bind in the DFG-out
orientation. The highly selective and potent nature of this
series gives it the potential to expand the mechanistic
understanding and application toward future therapies targeting
LIMK2.
56
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Letter
(11) Lamba, V.; Ghosh, I. New directions in targeting protein
kinases: Focusing upon true allosteric and bivalent inhibitors. Curr.
Pharm. Des. 2012, 18, 2936−2945.
(12) Hassan, A. Q.; Sharma, S. V.; Warmuth, M. Allosteric inhibition
of BCR-ABL. Cell Cycle. 2010, 9, 3710−3714.
(13) Gower, C. M.; Chang, M. C.; Maly, D. J. Bivalent inhibitor of
protein kinases. Crit. Rev. Biochem. Mol. Biol. 2014, 49, 102−115.
(14) Barf, T.; Kaptein, A. Irreversible protein kinase inhibitors:
Balancing the benefits and risk. J. Med. Chem. 2012, 55, 6243−6262.
(15) Bernard, O. Lim kinases, regulators of actin dynamics. Int. J.
Biochem. Cell Biol. 2007, 39, 1071−1076.
(16) Olson, M. F. Follow the leader: LIM kinases pave the way for
collective tumor cell invasion. Cell Cycle 2010, 9, 4417−4418.
(17) Harrison, B. A.; Whitlock, N. A.; Voronkov, M. V.; Almstead, Z.
Y.; Gu, K.; Mabon, R.; Gardyan, M.; Hamman, B. D.; Allen, J.;
Sopinathan, S.; McKnight, B.; Crist, M.; Zhang, Y.; Liu, Y.; Courtney,
L. F.; Key, B.; Zhou, J.; Patel, N.; Yates, P. W.; Liu, Q.; Wilson, A. G.
E.; Kimball, S. D.; Crosson, C. E.; Rice, D. S.; Rawlins, D. B. Novel
class of LIM-Kinase 2 inhibitors for the treatment of ocular
hypertension and associated glaucoma. J. Med. Chem. 2009, 52,
6515−6518.
(18) Huang, H.; Zhang, X.; Li, S.; Liu, N.; Lian, W.; McDowell, E.;
Zhou, P.; Zhao, C.; Guo, H.; Zhang, C.; Yang, C.; Wen, G.; et al.
Physiological levels of ATP negatively regulate proteasome function.
Cell Res. 2010, 20, 1372−1385.
(19) Gribble, F. M.; Loussouarn, G.; Tucker, S. J.; Zhao, C.; Nichols,
C. G.; Ashcroft, F. M. A novel method for measurement of
submembrane ATP concentration. J. Biol. Chem. 2000, 275, 20046−
20049.
(20) Increased concentrations of cold ATP in the LIMK2 assay
reduced the signal from hot ATP, resulting in unacceptable assay
performance. Because of the non-ATP competitive nature of these
compounds, this is not expected to alter the observed levels of
selectivity.
(21) A known type I LIMK2 inhibitor used as a reference was 1-(5-
methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-N-(3-phenoxyphenyl) piper-
idine-4-carboxamide and is described in ref 17.
(22) Cleland, W. W. Determining the chemical mechanisms of
enzyme-catalyzed reactions by kinetic studies. Adv. Enzymol. Relat.
Mol. Biol. 1977, 45, 273−387.
(23) Lineweaver, H.; Burk, D. The determination of enyzme
dissocation constants. J. Am. Chem. Soc. 1934, 56, 658−666.
(24) Cornish-Bowden, A. A simple graphical method for determining
the inhibition constants of mixed, uncompetitive, and non-competitive
inhibitors. Biochem. J. 1974, 137, 143−144.
57
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