Design of reversible, cysteine-targeted michael acceptors guided by kinetic and computational analysis

Article abstract of DOI:10.1021/ja505194w

Electrophilic probes that covalently modify a cysteine thiol often show enhanced pharmacological potency and selectivity. Although reversible Michael acceptors have been reported, the structural requirements for reversibility are poorly understood. Here, we report a novel class of acrylonitrile-based Michael acceptors, activated by aryl or heteroaryl electron-withdrawing groups. We demonstrate that thiol adducts of these acrylonitriles undergo β-elimination at rates that span more than 3 orders of magnitude. These rates correlate inversely with the computed proton affinity of the corresponding carbanions, enabling the intrinsic reversibility of the thiol-Michael reaction to be tuned in a predictable manner. We apply these principles to the design of new reversible covalent kinase inhibitors with improved properties. A cocrystal structure of one such inhibitor reveals specific noncovalent interactions between the 1,2,4-triazole activating group and the kinase. Our experimental and computational study enables the design of new Michael acceptors, expanding the palette of reversible, cysteine-targeted electrophiles.

Full text of DOI:10.1021/ja505194w

Article
pubs.acs.org/JACS
Open Access on 08/25/2015
Design of Reversible, Cysteine-Targeted Michael Acceptors Guided
by Kinetic and Computational Analysis
Shyam Krishnan,† Rand M. Miller,‡ Boxue Tian,§ R. Dyche Mullins,† Matthew P. Jacobson,§
and Jack Taunton*,†
†Department of Cellular and Molecular Pharmacology, Howard Hughes Medical Institute, University of CaliforniaSan Francisco,
San Francisco, California 94158, United States
‡Chemistry and Chemical Biology Graduate Program, University of CaliforniaSan Francisco, San Francisco, California 94158,
United States
§Department of Pharmaceutical Chemistry, University of CaliforniaSan Francisco, San Francisco, California 94158, United States
S
* Supporting Information
ABSTRACT: Electrophilic probes that covalently modify a cysteine thiol
often show enhanced pharmacological potency and selectivity. Although
reversible Michael acceptors have been reported, the structural require-
ments for reversibility are poorly understood. Here, we report a novel
class of acrylonitrile-based Michael acceptors, activated by aryl or
heteroaryl electron-withdrawing groups. We demonstrate that thiol
adducts of these acrylonitriles undergo β-elimination at rates that span
more than 3 orders of magnitude. These rates correlate inversely with the
computed proton affinity of the corresponding carbanions, enabling the
intrinsic reversibility of the thiol-Michael reaction to be tuned in a
predictable manner. We apply these principles to the design of new
reversible covalent kinase inhibitors with improved properties. A cocrystal
structure of one such inhibitor reveals specific noncovalent interactions
between the 1,2,4-triazole activating group and the kinase. Our experimental and computational study enables the design of new
Michael acceptors, expanding the palette of reversible, cysteine-targeted electrophiles.
targets.19 A major challenge in the field of covalent inhibitor
design is the identification of electrophiles that can form
INTRODUCTION
■
Chemical probes that bind covalently to a protein target often
energetically favorable yet reversible covalent bonds with
have prolonged target-residence times, resulting in increased
potency.1−3 Covalent probes can also be highly selective,
noncatalytic cysteines, which are often less nucleophilic than
catalytic cysteines.
especially when designed to react with a protein nucleophile
Activated acrylonitriles bearing a carboxylic ester or
that is not essential for enzymatic catalysis and poorly
conserved among closely related proteins.4 Examples of
carboxamide α-substituent have been reported to react rapidly
and reversibly with thiols.20,21 The thiol-Michael adducts could
covalent kinase inhibitors that target a noncatalytic cysteine
thiol include the BTK inhibitor, ibrutinib,5 and the EGFR
not be isolated and, upon dilution, underwent β-elimination to
inhibitor, afatinib,6 recently approved for advanced B cell and
form the starting cyanoacrylates/amides. Reversible thiol
reactivity appears to be a general property of cyanoacrylamides,
lung cancers, respectively. Both of these drugs employ an α,β-
and this insight led to the design of reversible, cysteine-targeted
unsaturated carboxamide to form an irreversible covalent bond
with a poorly conserved cysteine in the kinase active site.7
kinase inhibitors such as cyanoacrylamide 1 (Figure 1a), which
inhibits RSK1/2/4 kinases in the picomolar to low nanomolar
Despite their advantages, covalent drugs are seldom
range.21 Recently, we took advantage of the intrinsic
developed for diseases other than cancer because of the
reversibility of thiol/cyanoacrylamide reactions to develop an
electrophilic fragment-based approach to ligand discovery. This
strategy led to the first cysteine-targeted inhibitor of the MSK1
C-terminal kinase domain.22 The cyanoacrylamide inhibitors
exhibit slow off-rates when bound to the intact, folded kinase
domain, yet dissociate rapidly when the protein is unfolded or
potential for adverse effects caused by irreversible modification
of off-target nucleophiles.8−13 Exceptions to this trend include
older drugs whose covalent mechanism was initially unknown
(e.g., clopidogrel14 and omeprazole15), as well as recently
developed protease inhibitors that form reversible covalent
bonds with catalytically essential nucleophiles (e.g., telaprevir,16
odanacatib,17 and saxagliptin18). The latter compounds employ
weak electrophiles such as ketones or nitriles, which have not
found general utility outside the specific context of protease
Received: May 23, 2014
Published: August 25, 2014
© 2014 American Chemical Society
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different aryl or heteroaryl activating groups at the α-position
and a phenyl group at the β-position (Table 1). We then
measured β-elimination rates of the corresponding Michael
adducts derived from the simple thiol, β-mercaptoethanol
Table 1. β-Elimination Rates and Calculated Proton
Affinities of BME/Acrylonitrile Adducts 3a−9a
Figure 1. Reversible covalent binding of activated Michael acceptors to
cysteine thiols. (a) RSK2 inhibitors, 1 and 2. A cocrystal structure
(PDB code: 4D9U) reveals hydrophobic interactions between the tert-
butyl group of 2 and the glycine-rich loop of RSK2. (b) Reversible
covalent binding of an activated acrylonitrile to a hypothetical cysteine-
containing protein. In this example, the electron-withdrawing group
(EWG) and the β-substituent (R) both contribute to the free energy
of binding by providing favorable noncovalent interactions with the
protein.
degraded by proteases. As revealed by the cocrystal structure of
cyanoacrylate 2 bound to RSK2, the pyrrolopyrimidine scaffold
forms specific noncovalent interactions with the kinase,
orienting the electrophile and cooperatively stabilizing the
covalent complex. In addition, interactions between the tert-
butyl ester group and the glycine-rich loop of RSK2 likely
contribute to the slow off-rate.
The latter observation suggested the possibility of exploiting
the electron-withdrawing groups not only for their ability to
activate the olefin toward conjugate addition but also for their
potential to contribute specific noncovalent interactions
(Figure 1b). More generally, we recognized that, in many
targets, the orientation of the cysteine relative to the binding
site would be such that one of the olefin activating groups could
serve as the primary noncovalent recognition element. While
electrophilic olefins such as α-cyanoenones,23 alkylidene
rhodanines,24 and alkylidene thiazolidine diones25 have also
been found to form reversible covalent adducts with protein
thiols, little is known about the relationship between an
activated olefin’s electron-withdrawing substituents and its
propensity to react reversibly with thiols.26
RESULTS AND DISCUSSION
■
Kinetic and Computational Studies of Model Thiol/
Acrylonitrile Adducts. To expand the structural diversity of
reversible cysteine-targeted electrophiles, we replaced the
carboxamide group of 2-cyanoacrylamides with aryl or
heteroaryl activating groups. The nitrile group was retained
because of its small size and ability to form both polar and
hydrophobic interactions with proteins.27 At the outset, it was
not obvious how different aryl, or especially heteroaryl,
activating groups would affect the intrinsic reversibility of the
thiol-Michael reaction, i.e., the rate of thiol elimination from
acrylonitrile-derived Michael adducts. To our knowledge, no
systematic studies on the reactivity of such compounds toward
thiols have been reported. With the goal of establishing kinetic
trends that could be extrapolated to more complex structures,
we synthesized a series of model acrylonitriles (3−9) bearing
a
Determined by 1H NMR for adducts 3a−6a and 10a−11a after
dilution into DMSO-d6/PBS-d (3:1 v/v) and by LC-MS for adducts
7a−9a and 12a after dilution into PBS. Diastereomeric ratios of 3a−
6a, 8a, and 10a−11a remained constant after dilution, suggesting rapid
interconversion. The diastereomers of 7a and 12a were not separable
during LC-MS analysis. For 9a, a 1:1 ratio of diastereomers was
b
subjected to the β-elimination reaction. Proton affinities in water
(ΔGaq) were calculated for syn- and anti-diastereomers of thioethers
3a−12a using the B3LYP/6-311+G(d), IEFPCM method. ΔΔGaq
values relative to 3a are shown.
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(BME). These rates should reflect the relative propensity of α-
activated acrylonitriles to dissociate from cysteines in
unstructured regions of proteins (spurious off-target adducts)
or from unfolded globular proteins prior to their degradation by
cellular proteases.
After reacting the acrylonitriles (3−9) with excess BME, we
diluted the isolated adducts (7a−9a), or equilibrium mixtures
in the case of nonisolable adducts (3a−6a), into pH 7.4 buffer
(with DMSO cosolvent) to promote thiol elimination. β-
elimination rates were determined by monitoring the
disappearance of the adducts and reappearance of the
acrylonitriles by NMR or LC/MS (Supplementary Figures
S1−S10). Depending on the activating group, the half-times for
thiol elimination varied from less than 1 min to more than 2
days (Table 1 and Supplementary Table S1). Among the aryl/
heteroaryl activating groups tested, the methylthiazole (4a)
exhibited the greatest intrinsic reversibility (fastest elimination
rate, t1/2 < 1 min); the 3-pyridyl (7a) and 1,2,4-triazol-1-yl (8a)
adducts reverted to the starting acrylonitriles with intermediate
rates, whereas the pyrazol-1-yl adduct (9a) was essentially
irreversible (t1/2 > 58 h).
Figure 2. Brønsted-type plot of computed proton affinity (ΔΔGaq for
syn-diastereomers) vs β-elimination rate (log k, min−1) for BME/
acrylonitrile adducts 5a−12a (Table 1). Adducts 3a and 4a are not
shown, as only the upper limit of their t1/2 values (<1 min) could be
determined. BME/acrylonitrile adducts with higher proton affinity
(more negative ΔΔGaq) undergo β-elimination at slower rates.
Plotting ΔΔGaq for the anti-diastereomers affords a similar correlation
(Supplementary Figure S11).
the pKa of the thiolate leaving group, we purified and
characterized the cysteamine (pKa 8.4)36 adducts of triazole 8
and pyrazole 9. Upon dilution into pH 7.4 buffer, we found that
the β-elimination rates were 11.2- and 15.8-fold faster,
respectively, than the corresponding BME adducts (Supple-
mentary Figures S13 and S14). These results imply that more
intrinsically reactive surface cysteines (or cysteines in unfolded
regions of proteins), i.e., those with a lower pKa, are less likely
to form long-lived adducts with heteroaryl-acrylonitriles and
more likely to dissociate upon dilution or clearance of the
inhibitor. It is important to note, however, that we do not
anticipate any correlation between the elimination rates of
model thiol/acrylonitrile adducts (Table 1 and Figure 2) and
the potency of inhibitors bearing these electrophiles. This is
because potency will be affected by noncovalent interactions
(and clashes) between the acrylonitrile activating group and the
protein target, in addition to the rates of covalent bond
formation and dissociation with the active-site cysteine.
Application of Heteroaryl-Activated Acrylonitriles to
Covalent Kinase Inhibitors. We next sought to employ these
novel Michael acceptors in the design of reversible, cysteine-
targeted kinase inhibitors. We synthesized acrylonitriles 13−18,
all of which bear an aryl/heteroaryl activating group, analogous
to the model compounds in Table 1. A pyrrolopyrimidine
moiety attached to the electrophilic β-carbon serves as the
primary noncovalent recognition element (Figure 3a), analo-
gous to the cyanoacrylamide RSK2 inhibitors reported
previously.21 In addition to the activating groups studied
above (Table 1), we tested pyrazine 14 and pyridine 15 (Figure
3a), as they were both predicted to react reversibly with thiols
on the basis of their computed proton affinity (Supplementary
Table S2 and Figure S12).
To explore the potential for structural variation at the
electrophilic β-position, we tested three heteroaryl-activated
acrylonitriles (10−12) bearing a cyclopropyl in place of a
phenyl group (Table 1). Although elimination from the
cyclopropyl-substituted BME adducts was 2−3 times slower
than the corresponding phenyl-substituted adducts, the results
demonstrate that an aromatic substituent in the β-position is
not essential for reversibility of thiol-Michael reactions with
activated acrylonitriles. A smaller cyclopropyl group might be
preferred in reversible acrylonitrile-based ligands in which the
β-substituent is solvent-exposed and the α-activating group
serves as the primary noncovalent recognition element.
We used a computational approach to gain insight into the
structure/reactivity trends (Table 1), reasoning that increased
acidity of the α-carbon bearing the nitrile and the second
electron-withdrawing group would lead to faster thiol
elimination via an E1cB mechanism.28−31 Using density
functional theory, we calculated the proton affinities32 in
aqueous solution for the conjugate base (α-carbanion) of each
BME/acrylonitrile adduct (expressed as ΔΔGaq, relative to the
cyanoacrylamide-derived adduct 3a, Table 1). Similar trends
were observed using calculated proton affinities for either the
syn- or anti-diastereomers of the BME adducts. A Brønsted-type
plot33 revealed a linear correlation between the calculated
proton affinities and a three-log range of experimental rate
constants for thiol β-elimination (R2 = 0.96, Figure 2 and
Supplementary Figure S11). This combined computational/
empirical analysis suggests the possibility of predicting the
approximate rate of thiol elimination from adducts bearing
novel, as-yet-uncharacterized activating groups (Supplementary
Table S2), simply by calculating the proton affinity of the
corresponding α-carbanion (ΔGaq). To test this, we prepared a
new β-phenyl-substituted acrylonitrile bearing a pyrazine
activating group. The calculated ΔΔGaq for the derived BME
adduct is −3.4 kcal/mol; fitting this value to the Brønsted
relationship shown in Figure 2 led to a predicted β-elimination
t1/2 of 6.1 min. Using NMR spectroscopy, we determined the
t1/2 for BME β-elimination to be 7.2 min (k = 0.096 min−1),
close to the predicted value (Supplementary Figure S12).
BME has a pKa of 9.6,34 toward the high end of the range for
surface cysteines (from a recent study of surface cysteines:
mean pKa 9.3, range 8.2−9.9).35 To test the effect of decreasing
In kinase assays with the RSK2 C-terminal kinase domain,
pyrazine 14 was the most potent inhibitor (IC50 = 12 nM),
whereas the p-cyanophenyl compound 18 was the least potent
(IC50 = 770 nM). All of the inhibitors showed reduced potency
toward the Cys436 to Val mutant, consistent with covalent
bond formation with Cys436. We speculate that noncovalent
interactions contribute to the differential potency of com-
pounds 13−18, with thiazole 13 and pyrazine 14 having the
best steric and electrostatic complementarity to the RSK2
active site. To test whether acrylonitriles 13−18 bind RSK1/2
in cells, we used a competitive labeling assay with the
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Figure 3. Targeting RSK2 kinase with aryl/heteroaryl-activated acrylonitriles. (a) Half-maximal inhibitory concentration (IC50, mean s. d.) of
acrylonitriles 13−18 in kinase assays with wild-type RSK2 (WT) and the Cys436Val mutant (C436V). (b) Binding of acrylonitriles 13−18 (0.1 and
1.0 μM) and FMK (1.0 μM, positive control) to endogenous RSK1 and RSK2 in MDA-MB-231 breast cancer cells, assayed by competitive labeling
with the irreversible fluorescent probe, BODIPY-FMK. Cells were incubated with acrylonitriles 13−18 for 2 h prior to treatment with BODIPY-
FMK for 1 h. Cells were lysed and proteins were analyzed by gel electrophoresis, followed by fluorescence scanning and immunoblotting for RSK1/
2. (c) Dose response for RSK1/2 occupancy in MDA-MB-231 cells by 13 and 14 (RSK1/2 immunoblot: green and red respectively).
fluorescent affinity probe, BODIPY-FMK.21 Labeling of RSK1/
2 was significantly reduced in cells that were pretreated with
compounds 13−16, whereas compounds 17 and 18 were less
effective (Figure 3b). Thiazole 13 and pyrazine 14 were
especially potent, blocking endogenous RSK1/2 at low
nanomolar concentrations (EC50 ≈ 5 nM, Figure 3c).
develop potent inhibitors of the MSK1 C-terminal kinase
domain.22 These compounds also inhibit RSK2 and are thus
useful for interrogating signaling events downstream of the
closely related MSK/RSK subfamily of kinases in cellular assays
of short duration (<2 h). However, unlike our previously
developed pyrrolopyrimidine-based cyanoacrylamides,21 the
indazole-based cyanoacrylamides (e.g., compound 19, Figure
4) lose activity over a period of 12−24 h and are degraded in
cell culture by unknown mechanisms (unpublished results).
Remarkably, we were able to solve this problem by replacing
the carboxamide with a 1,2,4-triazole as the acrylonitrile
activating group. Although triazole 20 is somewhat less potent
than 19 in RSK2 kinase assays (IC50 = 47 vs 3 nM,
respectively), 20 has similar or better selectivity over NEK2
and PLK1, unrelated kinases with a homologous cysteine in the
ATP binding site (Figure 4a). Moreover, the cellular efficacy of
20 was dramatically enhanced, as shown by sustained RSK1/2
occupancy and the absence of compound degradation during a
24 h experiment. By contrast, RSK1/2 occupancy by
cyanoacrylamide 19 was lost after 24 h (Figure 4b), correlating
with its disappearance from the cell culture medium. Similar to
19, triazole 20 potently inhibited MSK1 autophosphorylation
in cells (Supplementary Figure S15) and had no significant
A desirable attribute of our previously described cyanoacry-
lamide inhibitors is their ability to dissociate from Cys436 (and
presumably any off-target thiols) after unfolding or proteolysis
of the intact RSK2 kinase domain.21,22 Our model studies with
activated acrylonitriles and BME (Table 1) suggested that
inhibitors 13−18 would share this property. To test this, we
incubated each inhibitor (1.6 μM) with excess RSK2 (3.2 μM),
resulting in quantitative formation of the complex based on the
kinase activity assays (Figure 3a). RSK2/inhibitor complexes
were then treated with 3 M guanidine (pH 7.4) to unfold the
kinase domain, and the released inhibitor was quantified by LC-
MS. Similar to our previously reported cyanoacrylamide
inhibitors, acrylonitriles 13−18 were recovered in 84−93%
yields after guanidine-mediated unfolding of the inhibitor-
bound RSK2 kinase domain (Supplementary Table S3).
We recently developed an electrophilic fragment-based
approach to ligand discovery, and we used this method to
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Figure 4. Potent, selective, and durable inhibition of RSK2 by a 1,2,4-triazole-activated acrylonitrile, 20. (a) Dose−response curves for 20 in kinase
assays with RSK2, NEK2, and PLK1 (IC50 values for compound 19 reproduced from ref 22). (b) RSK1/2 occupancy was determined 2 and 24 h
after treating cells with 19 and 20 (1 μM) by competitive labeling with BODIPY-FMK (RSK1/2 immunoblot: red and green, respectively). (c)
Overlay of 20/RSK2 cocrystal structure (this work) with NEK2 (red, PDB code: 2WQO), highlighting Leu546 of RSK2 and the structurally
homologous residue Phe148 of NEK2. The side chain of NEK2 Phe148 would clash with the 1,2,4-triazole.
effect when tested against a panel of 18 additional kinases, 10 of
which contain an active-site cysteine (Supplementary Table
S4). Despite the selectivity observed with this panel of cysteine-
containing kinases, we cannot rule out binding of 20 to other
cellular targets besides MSK/RSK. As expected on the basis of
our BME reactivity studies, binding of triazole 20 to RSK2 was
reversible upon unfolding the kinase domain (t1/2 ≈ 1 h at
room temperature), albeit with slower kinetics relative to 19. By
contrast, the corresponding pyrazole-acrylonitrile S3 was
essentially irreversible in this assay, showing ∼2% dissociation
after 4 h (Supplementary Figure S16).
A cocrystal structure of triazole 20 bound to RSK2 (PDB:
4M8T) confirmed the covalent bond with Cys436 and revealed
specific noncovalent interactions between the trimethoxyphenyl
indazole scaffold and the kinase active site (Figure 4c). The
1,2,4-triazole projects from the newly formed sp3-carbon
toward the floor of the ATP binding site, forming close
contacts (4−5 Å) with the methyl groups of Leu546. The
analogous region of both PLK1 and NEK2 is occupied by a
larger phenylalanine residue, which would likely clash with the
triazole. Thus, despite sharing a homologous cysteine, PLK1
and NEK2 are apparently unable to accommodate the triazole
upon nucleophilic attack and protonation of the acrylonitrile.
The 1,2,4-triazole substituent in 20 serves three critical
functions: (1) it activates the acrylonitrile toward reversible
nucleophilic attack by Cys436; (2) it enhances selectivity via
polar and hydrophobic interactions with RSK2 and a steric
clash with PLK1 and NEK2; and (3) it increases cellular
stability and potency.
CONCLUSIONS AND PERSPECTIVE
■
In this study, we have characterized a series of aryl- and
heteroaryl-activated acrylonitriles, with the ultimate goal of
applying these novel electrophiles to the design of reversible,
cysteine-targeted probes. We focused our attention on the
kinetic stability of the corresponding thiol-Michael adducts,
reasoning that electrophiles with greater intrinsic reversibility
(i.e., giving rise to kinetically less stable thiol adducts) are less
likely to form permanent covalent adducts with off-target
cysteines.9 As with irreversible covalent inhibitors, the
maximum residence time of reversible covalent inhibitors is
limited by the turnover rate of the target. Hence, the reversible
covalent strategy would have a limited pharmacodynamic
advantage when targeted against a protein with a short half-life,
although the selectivity advantage gained by targeting a
noncatalytic cysteine would still apply. High intrinsic
reversibility may also be advantageous in electrophilic frag-
ment-based screens, although a recently published screen of
irreversible acrylate-based fragments suggests that reversible
electrophiles may not be required.37 With intrinsically
reversible electrophiles, fragment binding would be under
thermodynamic control and would require specific noncovalent
interactions to cooperatively stabilize a high-affinity covalent
interaction with the target.22 Our DFT calculations on thiol/
acrylonitrile adducts and their derived carbanions revealed a
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experimental β-elimination rates. Based on this analysis, we can
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Detailed experimental procedures, synthesis and spectral
characterization of compounds, kinetic measurements, compu-
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parameters. This material is available free of charge via the
Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
jack.taunton@ucsf.edu
■
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The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
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This work was supported by grants from the U.S. National
Institutes of Health (NIH) (GM071434 to J.T.), the California
Tobacco Related Disease Research Program (19FT-0091 to
S.K.), and the National University of Ireland-Galway (B.T.).
We acknowledge the University of CaliforniaSan Francisco
(UCSF) Mass Spectrometry Facility (supported by NIH Grant
P41RR001614), the SFI/HEA Irish Centre for High-End
Computing (ICHEC), and Prof. Leif A. Eriksson (University of
Gothenburg) for helpful discussions.
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