Predicting the reactivity of nitrile-carrying compounds with cysteine: A combined computational and experimental study

Article abstract of DOI:10.1021/ml400489b

Here, we report on a mechanistic investigation based on DFT calculations and kinetic measures aimed at determining the energetics related to the cysteine nucleophilic attack on nitrile-carrying compounds. Activation energies were found to correlate well with experimental kinetic measures of reactivity with cysteine in phosphate buffer. The agreement between computations and experiments points to this DFT-based approach as a tool for predicting both nitrile reactivity toward cysteines and the toxicity of nitriles as electrophile agents.

Full text of DOI:10.1021/ml400489b

Letter
pubs.acs.org/acsmedchemlett
Predicting the Reactivity of Nitrile-Carrying Compounds with
Cysteine: A Combined Computational and Experimental Study
Anna Berteotti,† Federica Vacondio,‡ Alessio Lodola,‡ Michele Bassi,‡ Claudia Silva,‡ Marco Mor,*,‡
and Andrea Cavalli*,†,§
†Drug Discovery and Development, Italian Institute of Technology, via Morego 30, 16163 Genova, Italy
‡Dipartimento di Farmacia, Universita
̀
degli Studi di Parma, Parco Area delle Scienze 27/A, 43124 Parma, Italy
§Department of Pharmacy and Biotechnology, University of Bologna, via Belmeloro 6, 40126 Bologna, Italy
S
* Supporting Information
ABSTRACT: Here, we report on a mechanistic investigation
based on DFT calculations and kinetic measures aimed at
determining the energetics related to the cysteine nucleophilic
attack on nitrile-carrying compounds. Activation energies were
found to correlate well with experimental kinetic measures of
reactivity with cysteine in phosphate buffer. The agreement
between computations and experiments points to this DFT-
based approach as a tool for predicting both nitrile reactivity
toward cysteines and the toxicity of nitriles as electrophile agents.
KEYWORDS: Cysteine, nucleophilic attack, DFT, nitrile reactivity
Nitriles can react with serine or cysteine residues of proteases
itriles are one of the most common chemical groups in
Nnature. They are versatile synthetic intermediates and
important compounds per se.1 They are endowed with rich
chemistry and serve as precursors for various functional groups,
such as amines, amidines, tetrazoles, aldehydes, amides, and
other carboxy derivatives.2 They are also key motifs in
numerous compounds of practical utility including dyes,
herbicides, agrochemical, electronic materials, and drugs.3,4 In
drug discovery, a deep interest in nitrile-containing pharma-
ceuticals is emerging, as shown by the high number (about 30)
of nitrile-carrying drugs that are currently in use for a variety of
pathological conditions.5 In addition, more than 20 nitrile-
containing drug candidates are currently in clinical develop-
ment. The emerging importance of nitrile in this field is due to
its remarkable versatility. This is related to the short, polarized
triple bond,6 which allows nitrile-containing molecules to have
a large variety of different types of interactions. Several crystal
structures of nitriles in complex with biological targets show the
nitrile projecting into narrow clefts to make polar interactions
or hydrogen bonds in sterically congested environments.7 They
can also play a key role as hydrogen-bond acceptors. Several
crystal structures show hydrogen bonding between the nitrile
nitrogen and amino acids or water-mediated interactions with
protein backbones.8 In other cases, the strong dipole facilitates
polar interactions, in which the nitrile acts as a hydroxyl or
carboxyl isostere.9 Most nitrile-containing pharmaceuticals are
aromatics with aliphatic-, alkene-, and nitrogen-bound nitriles
(cyanamides) being progressively less frequent. Very well-
known examples of nitrile-containing pharmaceuticals are the
inhibitors of aromatase, such as Anastrazole10 or Letrozole,11
used for the treatment of estrogen-dependent breast cancer.
to afford an imidate or thioimidate covalent adduct,
respectively.12 The use of a nitrile group as a warhead has
gained considerable importance in covalent drug discovery,13 as
this functional group is chemically less reactive than aldehydes.
Several nitrile derivatives have been developed as reversible
covalent modifiers of serine and/or cysteine proteases,
including DPP-IV14 and cathepsin inhibitors15 for the treat-
ment of type 2 diabetes and osteoporosis. In the first case, the
inhibitors (e.g., vildagliptin16) covalently interact with the active
site Ser610 residue, forming a reversible covalent imidate
adduct. Similarly, a search for cathepsin K inhibitors led to a
series of amidoacetonitrile inhibitors, in which the nitrile
participates in a reversible, covalent interaction with the active
site cysteine residue.17
There are three known chemical classes of nitrile-containing
covalent inhibitors: (i) heteroaromatic nitriles, (ii) cyanamides,
and (iii) amidoacetonitriles.5,18 Despite their different electro-
philicity, all three classes can reversibly react with biological
thiols (including glutathione and proteins) giving a thiomidate
intermediate according to a chemical process resembling the
Pinner reaction. Moreover, when the thiol group belongs to a
cysteine with a free amino group, the readily formed thiomidate
rapidly evolves into a thiazoline product (Scheme 1), whose
chemical stability makes the overall reaction an irreversible
process.18,19
Received: November 28, 2013
Accepted: February 24, 2014
Published: February 24, 2014
© 2014 American Chemical Society
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Scheme 1. Nitrile-Containing Compounds Reacting with a
a
Cysteine Residue
a
The nitriles react with cysteine residue forming a thioimidate
intermediate that readily evolves into a thiazoline product.
Computational tools20 and/or experimental measures can be
used to predict reactivity toward biological targets and toxicity
toward off-targets. This is emerging as a key strategy in the
discovery of novel nitrile-containing drugs.18,21,22 Oballa et al.18
calculated the theoretical reactivities of structurally diverse
nitriles as the energy differences between the thioimidate
adduct, a precursor thiol nucleophile, and the parent nitriles. In
MacFaul et al.,21 the authors reported on an in vitro assay for
assessing the reactivity of nitrile-containing compounds toward
glutathione (GSH) and cysteine. In Ehmke et al.,22 the use of
density functional theory (DFT) calculations provided relative
reactivities of the nitriles and helped predict their biological
affinity and cytotoxicity.
Figure 1. (A) Reaction mechanism using as a representative case
benzonitrile. (B) PES as a function of IRC progression. (C,D) The
progression along the IRC of the main geometrical features that
change along the reaction.
In both Oballa’s and MacFaul’s studies,18,21 reaction with free
cysteine was used as an experimental model to probe nitrile
reactivity. Actually, this system is only representative of
reactions in biological environments where a thiolate attack is
assisted by a proton transfer mechanism. This mechanism is
found in many cysteine proteases, and particularly with Ntn-
hydrolase,23 where a terminal cysteamine-like fragment acts
both as a nucleophile and general acid.
Eleven different nitrile-carrying molecules were investigated
comprising aliphatic, aromatic, and heteroaromatic species. In
addition, three nitrile-containing pharmaceutical compounds
(i.e., one drug, one clinical candidate, and one pharmacological
tool) were studied using the same computational and
experimental approaches.
In Figure 1, we report a schematic representation of the
reaction here investigated (Figure 1A), the potential energy
surface (PES) associated with the reaction (Figure 1B), and the
evolution of some geometrical parameters as functions of the
IRC (Figures 1C,D). Starting from the reactants, the sulfur
atom of the cysteamine approached the carbon atom of the
nitrile group: the distance S−C2 decreased, while H1 moved
toward the nitrogen atom of the nitrile (see distance H1−N2 in
Figure 1C). The hybridization of the nitrile carbon changed
from sp to sp2 during the reaction. This could be deduced by
observing the C2−N2 distance, which increased, whereas the
C1−C2−N2 angle decreased (Figure 1D). The reaction
occurred through a concerted synchronous mechanism. At
the TS, the nucleophilic attack and the protonation happened
simultaneously. Figure 1C shows that starting from the
reactants the S−C2 and H1−N2 distances decreased along the
IRC pathway.
Here, we present a combined computational and exper-
imental study aimed at assessing the predictive power of a
DFT-based tool (B3LYP/6-311++G(d,p)) in studying nitriles
as covalent binders to cysteine residues. In previous studies, the
electrophilicities were estimated from the formation enthalpies
of the thioimidate adduct, as the product of the reaction
between methanethiol and nitriles. Then, the calculated
enthalpies were used for qualitative correlations with
experimental kinetic data. Although these approaches could
provide meaningful information, they were based on compar-
isons between thermodynamic and kinetic entities. In the
present study, activation energy (Ea) values, rather than
enthalpy differences, were compared to kinetic experimental
measures. In particular, we investigated the reaction mechanism
of thioimidate formation (Scheme 1) and compared theoretical
and experimental results.
As a model system for the DFT calculations, we used
cysteamine in its zwitterionc form (Figure 1A), which leads to a
thioimidate as the product of the nucleophilic attack on a
nitrile. All the simulations were carried out in implicit water.
We first identified and characterized the transition state (TS)
structures, and then we investigated the intrinsic reaction
coordinate (IRC) pathways that lead to the reactants (R) and
to the product (P). In this way, we calculated the Ea going from
R to TS. In parallel, the experimental reactivity of the nitriles
with a large excess of cysteine was studied by means of HPLC−
UV and HPLC−ESI−MS measures, monitoring the decreasing
concentration of the nitrile compounds as the reaction
proceeded at 37 °C (see Supporting Information). The
reactivity of a subset of nitriles was also measured with
cysteamine and GSH by HPLC−UV and HPLC−ESI−MS/
MS. These data are reported in the Supporting Information
(Figures S1 and S2).
As shown in Figure 2, the TS displayed a ring structure
assuming a chair-like conformation. This chair-like conforma-
tion was remarkably similar to those previously reported for
Figure 2. Transition state (TS) structure for benzonitrile 6 reacting
with cysteamine. A chair-like structure composed of a 7-membered
ring can be identified.
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similar reactions in enzymatic environment.23−25 Furthermore,
the concerted nature of the reaction mechanism here studied
was also in good agreement with the amide hydrolysis catalyzed
by classic cysteine hydrolases, such as papaine25 or cathepsin
K.26
Table 2. Experimental t1/2 (min), Standard Deviation (SD),
and Theoretical Activation Energy (Ea, kcal/mol) for Nitriles
12−14
All the nitrile-carrying molecules here investigated (see Table
1) and the three nitrile-carrying pharmaceutical compounds
Table 1. Experimental t1/2 (min), Standard Deviation (SD),
and Theoretical Activation Energy (Ea, kcal/mol) for Nitriles
1−11
Figure 3. Correlation between the Ea calculated as the difference
between the reactants and TSs and the experimental log(k). The black
diamonds report the data from the compounds 1−11, while the red
squares show the data for the three pharmaceutical compounds (12−
14).
reported on a log scale. As expected, pyrimidine, pyrazine, and
pyridine nitriles (compounds 1−5 in Table 1) were highly
reactive toward cysteine, due to the electron-withdrawing effect
of the heteroaromatic rings linked to nitriles. The 2-
cyanopyridine with a bromine atom in the para position (5)
showed higher electrophilicity relative to the unsubstituted
cyanopyridine; this can be attributed to the electron-with-
drawing effect of the bromine atom. The benzonitrile (6) and
the 4-chlorobenzonitrile (7) showed intermediate reactivities
toward cysteine. When the benzonitrile was substituted with an
electron donor amino group (8), the molecule was far less
reactive; this was due to the electron donor effects of the
substituent in position 4. The aliphatic nitriles (9 and 10)
showed an electrophilicity, which was between that of the
benzonitrile substituted in para with electron donor groups and
the more electron-deficient heteroaromatic nitriles. Finally, the
acetonitrile (11) was more reactive than benzonitriles 6−8 and
less reactive than pyrimidine, pyrazine, and pyridine nitriles 1−
5. Figure 3 also shows that computational data and
(see Table 2) showed remarkably similar reaction mechanisms,
which are reported in the Supporting Information. From our
calculations, we estimated reactivity of nitriles from the Ea
values, and we correlated these values to the experimental
kinetic constants of the nitriles reacting with cysteine in
phosphate buffer.
Among the most abundant ion species of cysteine at pH
7.4,27,28 the zwitterionic thiolate (depicted in Scheme 1) is
probably the most reactive one. Under this hypothesis and
considering the contribution of the carboxylate group as a
constant, we used a cysteamine in the calculations as a
simplified model of a cysteine.
In Figure 3, we plotted the calculated Ea versus the
experimental second-rate kinetic constants (k in M−1 min−1)
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experimental measures correlated well, with an R2 value of 0.86
(s = 1.14 and F = 54.1), considering compounds 1−11.
Increasing the complexity of the structure, as with 12−14,
decreased the predictive power of our DFT calculations. The
reason for this limited predictivity is probably multifactorial as it
depends both on some aproximations of B3LYP and on the use
of static geometries, which do not take into account how costly
it can be achieving the reactive conformation leading to the
transition state.29
We finally performed additional single-point calculations at a
higher level using the coupled-cluster theory with single and
double excitations (CCSD). CCSD calculations provided the
best correlation between theory and experiment (Figure S1,
Supporting Information), with an R2 of 0.88 (s = 1.06 and F =
63.9). However, the computational cost of this method
increases very rapidly with the number of atoms, which could
restrict its application to quite small molecules. From a drug
design perspective, DFT calculations, which are faster than
CCSD ones, may be a good compromise between speed and
accuracy.
The Ea values calculated by our approach can be considered
as a relative scale expressing the propensity of a nitrile group, in
a certain chemical environment, to react with thiol
nucleophiles. Our computations are substantiated by experi-
ments. They show that when the Ea is much less than 16 kcal/
mol, nitriles can react with the cysteine on a small time scale,
showing a reactivity similar to that of activated aromatic nitriles
or aminoacetonitriles. These are generally considered to be
covalent binders to biological targets. Conversely, when the Ea
value approached or overcame 20 kcal/mol (e.g., compounds 8
and 10), the cysteine nucleophilic attack was extremely slow in
our experimental conditions, with a half-life for nitrile
disappearance that could be higher than 20 h, as observed for
aliphatic nitriles or aromatic ones with conjugated electron-
donating groups. The present DFT-based tool and the
threshold value here reported could be used to predict how
promptly a nitrile will react with a cysteine, leading to a
covalent adduct. This could be extremely helpful in covalent
drug design and for predicting possible off-target liabilities for
nitrile-carrying drug candidates.
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ASSOCIATED CONTENT
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AUTHOR INFORMATION
■
Corresponding Authors
*(M.M.) E-mail: marco.mor@unipr.it.
*(A.C.) E-mail: andrea.cavalli@unibo.it.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Dr. Philip MacFaul (AstraZeneca) is kindly acknowledged for
providing compound 12 (Balicatib). The authors acknowledge
the IIT Platform “Computation” for computational resources.
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