Photogeneration of Quinone Methides as Latent Electrophiles for Lysine Targeting

Article abstract of DOI:10.1021/acs.joc.8b01559

Latent electrophiles are nowadays very attractive chemical entities for drug discovery, as they are unreactive unless activated upon binding with the specific target. In this work, the utility of 4-trifluoromethyl phenols as precursors of latent electrophiles, quinone methides (QM), for lysine-targeting is demonstrated. These Michael acceptors were photogenerated for specific covalent modification of lysine residues using human serum albumin (HSA) as a model target. The reactive QM-type intermediates I or II, generated upon irradiation of 4-trifluoromethyl-1-naphthol (1)@HSA or 4-(4-trifluorometylphenyl)phenol (2)@HSA complexes, exhibited chemoselective reactivity toward lysine residues leading to amide adducts, which was confirmed by proteomic analysis. For ligand 1, the covalent modification of residues Lys106 and Lys414 (located in subdomains IA and IIIA, respectively) was observed, whereas for ligand 2, the modification of Lys195 (in subdomain IIA) took place. Docking and molecular dynamics simulation studies provided an insight into the molecular basis of the selectivity of 1 and 2 for these HSA subdomains and the covalent modification mechanism. These studies open the opportunity of performing protein silencing by generating reactive ligands under very mild conditions (irradiation) for specific covalent modification of hidden lysine residues.

Full text of DOI:10.1021/acs.joc.8b01559

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Article
Photogeneration of Quinone-Methides as
Latent Electrophiles for Lysine-Targeting
Raul Perez-Ruiz, Oscar Molins-Molina, Emilio Lence, Concepción
González-Bello, Miguel A Miranda, and M. Consuelo Jiménez
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b01559 • Publication Date (Web): 02 Oct 2018
Downloaded from http://pubs.acs.org on October 6, 2018
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Photogeneration of Quinone-Methides as Latent Electrophiles for
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a, b
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c
Raúl PérezꢀRuiz, Oscar MolinsꢀMolina, Emilio Lence, Concepción GonzálezꢀBello, * Miguel A.
a
a
Miranda, * M. Consuelo Jiménez *
a
Departamento de Química/Instituto de Tecnología Química UPVꢀCSIC, Universitat Politécnica de València, Camino de
Vera s/n 46071 Valencia (Spain).
b
Photoactivated Processes Unit, IMDEA Energy Institute, Av. Ramón de la Sagra 3, 28935 Móstoles, Madrid (Spain).
Centro Singular de Investigación en Química Biolóxica e Materiais Moleculares (CIQUS), Departamento de Química Orꢀ
c
gánica, Universidade de Santiago de Compostela, Jenaro de la Fuente s/n, 15782 Santiago de Compostela (Spain).
ABSTRACT: Latent electrophiles are nowadays very attractive chemical entities for drug discovery, as they are unreactive unless
activated upon binding with the specific target. In this work, the utility of 4ꢀtrifluoromethyl phenols as precursors of latent electroꢀ
philes − quinone methides (QM) − for lysineꢀtargeting is demonstrated. These Michael acceptors were photogenerated for specific
covalent modification of lysine residues using human serum albumin (HSA) as a model target. The reactive QMꢀtype intermediates
I or II, generated upon irradiation of 4ꢀtrifluoromethylꢀ1ꢀnaphthol (1)@ HSA or 4ꢀ(4ꢀtrifluorometylphenyl)phenol (2)@HSA comꢀ
plexes, exhibited chemoselective reactivity towards lysine residues leading to amide adducts, which was confirmed by proteomic
analysis. For ligand 1, the covalent modification of residues Lys106 and Lys414 (located in subꢀdomains IA and IIIA, respectively)
was observed, whereas for ligand 2, the modification of Lys195 (in subꢀdomain IIA) took place. Docking and molecular dynamics
simulation studies provided an insight into the molecular basis of the selectivity of 1 and 2 for these HSA subꢀdomains and the
covalent modification mechanism. These studies open the opportunity of performing protein silencing by generating reactive ligꢀ
ands under very mild conditions (irradiation) for specific covalent modification of hidden lysine residues.
xenobioticꢀinduced liver and lung toxicity emerged, as well as
the idea that drugꢀprotein adducts are potential haptens able to
INTRODUCTION
Small organic molecules can undergo bioactivation in vivo,
which affords electrophilic species able to react with biomꢀ
acromolecules, leading to covalent adducts that trigger undeꢀ
sired toxic effects. This was already described in the 30’s for
the metabolites of polycyclic aromatic hydrocarbons, which
were found to be highly reactive intermediates able to attach to
elicit immuneꢀmediated adverse events. Such irreversible
binding was viewed as a risk factor in drug development,
either by the direct tissue damage or by the immune responses
after protein haptenation. However, the last decade has witꢀ
nessed the renaissance of targeted covalent inhibition in drug
2
discovery, for example in the successful treatment of cancer
by covalent epidermal growth factor receptor (EGFR) inhibiꢀ
1
liver proteins. During the 70s, the covalent binding theory of
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tors. The enormous advantages of irreversible drugs are assoꢀ
ciated with their high potency, low dosage, extended duration
trifluoromethylbenzoic acid and the εꢀamino groups of the
lysine residues of ubiquitin under sunlight exposure has been
recently demonstrated by photophysical and proteomic analyꢀ
4
of action, selectivity and general applicability. These features,
20
together with an improved understanding of their potential
risks, have resulted in a remarkable increase of the number of
clinical trials, drug approvals and scientific articles in the
sis.
Scheme 1. Targeted Irreversible Inhibition Approaches
4
area.
In this context, binding and reactivity are the two key steps
involved in “protein silencing”. The former consists in the
reversible association between an inhibitor (a highꢀaffinity
ligand) and its biological target, whereas the latter is the reacꢀ
tion between both partners to form a covalent adduct. Inhibiꢀ
tors of this type usually contain an electrophilic functional
group that reacts with a close nucleophile on the target. Their
design is highly challenging because it requires an appropriate
combination of affinity, reactivity and selectivity, to avoid offꢀ
target effects. Hence, electrophiles that are unreactive unless
activated upon protein binding (latent electrophiles, LEs) are
of particular interest in drug development. This constitutes an
attractive research field, as only few selective and safe LEs
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To check whether photogenerated QMs can be appropriate
LEs for lysineꢀtargeting, we selected 4ꢀtrifluoromethylꢀ1ꢀ
naphthol (1) and 4ꢀ(4ꢀtrifluorometylphenyl)phenol (2) (Chart
1
) as model compounds. The naphthalene and biphenyl chroꢀ
mophores should be advantageous for spectroscopic measureꢀ
ments. As regards the model protein, human serum albumin
(
HSA) was chosen taking into account its availability and dark
21
5
binding capability. The obtained results clearly show that
formation of groundꢀstate complexes between ligands and
HSA leads ultimately to stable adducts between photogeneratꢀ
ed LEs and lysine residues localized in the binding pockets of
HSA.
have been reported.
Strategically, targeted covalent inhibition requires the catalytic
action of a protein to convert a ligand into the corresponding
LE within the active site (Scheme 1, pathway A). Although
this is now considered as a validated tool for drug discovery,
alternative strategies for addressing this issue would be desiraꢀ
ble. One interesting possibility could be the utilization of light
as an external factor to achieve direct formation of LEs from
the bound ligand, without the need for any catalytic process
mediated by the protein (Scheme 1, pathway B).
Chart 1. Chemical structures of 1 and 2
OH
HO
CF
3
CF3
To inactivate proteins through formation of irreversible adꢀ
ducts, alkylations or Michael additions are among the most
frequently employed reactions.
methides (QMs) are good Michael acceptors that can react
with the amino acid residues of proteins (commonly Cys) and6
other QM intermediates have been found to modify proteins;
in this context QM have been applied as suitable LEs for the
inhibition of mammalian serine proteases and bacterial serine
1
2
4
c,d
Specifically, quinone
RESULTS AND DISCUSSION
The methodological approach to achieve the proposed objecꢀ
tive involved i) characterization of the excited states of 1 and 2
in solution, both as the free compounds and in the presence of
proteins, ii) proteomic studies of 1 and 2 irradiated in the
presence of HSA, to detect covalent photobinding to amino
acid residues and iii) theoretical calculations, to understand the
binding modes and the formation of adducts. The obtained
results are presented below in separated sections.
7
8
βꢀlactamases. However, the photogeneration of QMs within
proteins has been rarely reported, despite the possibility of
exciting selectively their precursors under mild conditions.
9
Trifluoromethyl substituted aromatic moieties are present in a
number of agrochemicals, polymers and pharmaceuticals.
In general, incorporation of a trifluoromethyl substituent enꢀ
hances the lipophilicity of active compounds and results in a
better incorporation into target cells. In comparison with other
halogenꢀcontaining compounds, trifluoromethyl derivatives
are less reactive
However, a number of trifluoromethyl aromatics can be conꢀ
verted into carboxylic acid derivatives in the presence of nuꢀ
cleophiles upon photochemical activation. In this regard, the
photohydrolysis of trifluoromethylꢀsubstituted phenols and
naphthols has been previously reported to occur through forꢀ
1
0
11
12
Photophysical detection of a complex between trifluoro-
methylphenols 1 and 2 and HSA. First, the absorption and
emission properties of 1 were determined in acetonitrile and in
aqueous media, under neutral and alkaline conditions (pH =
13,14
15
and reluctant to biological degradation .
7
.4 or pH=12), to ensure predominance of the phenol or the
phenolate anion in the ground state. A summary of the obꢀ
tained results is presented in Table 1. It was found that the free
phenol absorbs at ca. 300 nm and emits at ca. 340 nm, whereꢀ
as the corresponding bands of the phenolate displayed maxima
at ca. 340 and 450 nm, respectively.
1
6
1
7
mation of QM intermediates.
In the case of 2, the free phenol showed absorption and emisꢀ
sion peaking at 270ꢀ275 nm and 350ꢀ365 nm, respectively.
The corresponding bands of the phenolate were found at 305
and 430 nm. Some representative spectra of 1 and 2 under
different conditions are shown in Figures S1ꢀS3 of the Supꢀ
porting Information.
With this background, we decided to explore the feasibility of
the targeted covalent inhibition concept through photochemiꢀ
cal generation of LEs and subsequent reaction with targeted
protein nucleophiles. Compared to cysteine residues, targetꢀ
ing nucleophilic lysine residues of the bindingꢀsites appears
18
19
more challenging and has been less exploited. In this context,
identification
of
adducts
between
2ꢀhydroxyꢀ4ꢀ
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Table 1. Photophysical properties of 1 and 2
B
Compound
Medium
(λ
)
/nm
(λ ) /nm
max abs
max em
1
MeCN
pH = 7.4
pH = 12
HSA
295
301
343
342
446
447
420
2
50
300
350
400
λ / nm
327,373
0
3
50
400
450
500
550
600
650
2
MeCN
pH = 7.4
pH = 12
HSA
275
270
306
315
348
364
430
415
λ / nm
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1,0
C
0
,8
,6
0
When the absorption spectrum of 1 was recorded in PBS, in
the presence of HSA, a new band emerged above 320 nm,
which increased with increasing concentrations of protein. A
similar trend was observed for 2 in the presence of HSA, altꢀ
hough the new band was more intense compared to that of 1
(Figures S4A,B). In both cases, the new bands were attributed
to a groundꢀstate complex with HSA. Selective excitation of
the complexes led to new emission bands centered at λ = 420
nm (1) or λ = 415 nm (2), which again increased with increasꢀ
ing concentrations of protein (Figure S4C,D) and were differꢀ
ent from those previously ascribed to the corresponding pheꢀ
nol or phenolate (Figure 1A,B); they were attributed to emisꢀ
sion from the above mentioned HSA complexes. Control
experiments, consisting on excitation of HSA alone, indicated
the absence of emission arising from the protein under the
experimental conditions (Figure S5). The stoichiometry of the
1@HSA and 2@HSA complexes was determined as 2:1 and
0,4
0,2
0,0
350
400
450
500
550
600
650
λ/ nm
Figure 1. Emission (large) and absorption (inset) spectra, recordꢀ
ed in PBS (red) and in the presence of 2 equivalents of HSA
(blue) for 1 (A) and 2 (B), respectively; λexc was 355 nm for 1 and
3
24 nm for 2. (C): Normalized transient absorption spectra obꢀ
tained for 1 (orange) and 2 (black) in PBS in the presence of 5
equivalents of HSA, upon laser excitation at 355 nm. In all measꢀ
urements, [1] = 0.05 mM, [2] = 0.05 mM
Laser flash photolysis of the 1@HSA and 2@HSA
complexes was also performed upon excitation at 355 nm. In
both cases, the resulting transient absorption spectra (Figure
C) were tentatively ascribed to the expected quinone methiꢀ
des I and II (Chart II), based on the existing data on related
1
1
:1, respectively, by means of Job plot analysis (SI, Figure
22
S6). To evidence formation of 1@HSA and 2@HSA comꢀ
plexes, difference spectra, i. e. [1@HSA]ꢀ[1+HSA], were
obtained upon variations of ligand/protein molar ratios (Figꢀ
ures S7A and S8A). To calculate the binding constants, the
compounds. Their lifetimes were 0.5 and 8.0 ꢁs, respectiveꢀ
8h,i
ly.
Photobinding of 1 and 2 to HSA. Irradiation of 1 and 2 in
aqueous media (λmax = 300 nm) led to the corresponding carꢀ
boxylic acids, in agreement with the previously reported result
for similar substrates. The accepted mechanism involves
deprotonation in the singlet excited state, followed by heteroꢀ
lytic CꢀF bond cleavage to afford a QMꢀtype intermediate (I or
23
BenesiꢀHildebrand procedure (eq. 1, Figures S7B and S8B)
was employed.
ꢀ
ꢁꢂꢃꢄꢅꢆꢇ
ꢒꢓ
ꢒ
= ꢔ
+ ɛ
Eq. 1º
ꢈꢉꢊꢋꢌꢍꢎꢏꢐꢑ
ꢋꢌꢍꢎꢏꢐꢑꢓ×ꢀꢕꢖꢈꢇ×ɛꢋꢌꢍꢎꢏꢐꢑ
ꢋꢌꢍꢎꢏꢐꢑ
17
II, Chart 2).
Photolysis of 1@HSA and 2@HSA was performed
for detecting the possible formation of covalent photoadducts
([ligand] = 5 × 10 M; ligand:protein 1:5 molar ratio, air/PBS,
where Abscomplex is the maximum absorbance of complexes (at
73 nm for 1 and 315 nm for 2) at different HSA concentraꢀ
tions and εcomplex is the molar absorption coefficient. The hhgꢀ
affinity K values, determined from the slope, are 2.4 ×
3
−5
λmax irr. = 300 nm). The photodegradation process was moniꢀ
tored by fluorescence spectroscopy (Figure S9). Then, trypsin
digestion (to cleave peptide chains mainly at the carboxyl side
of Lys or Arg residues, unless there is a neighboring Pro resiꢀ
due) was run, coupled with HPLCꢀMS/MS analysis. Fullꢀscan
and fragmentation data files were treated by using the Masꢀ
cotS database search engine, in order to find out which (if any)
of the Lys residues located inside the hydrophobic binding
sites of the protein became covalently modified by nucleoꢀ
philic trapping of intermediates I or II (Chart 2).
complex
5
ꢀ1
4
ꢀ1
1
0 M and 2.8 × 10 M , for 1 and 2, respectively. These
results are consistent with data previously reported for similar
2
4
compounds such as naphthol and hydroxybiphenyl.
4000
2
A
1
0
2
50
300
350
400
λ
/ nm
Chart 2. Chemical structures of the QM intermediates I and II
together with the Lys trapping products
0
400
450
500
λ / nm
550
600
650
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O
OH
C
Lys106 in subꢀdomain IA were used to define the recognition
site, and the radius was set to 8 Å. The highest score solutions
obtained by docking were further analyzed by MD simulation
studies in order to assess the stability and therefore the reliaꢀ
bility of the postulated binding. The monomer of the 1@HSAꢀ
IA and 1@HSAꢀIIIA protein complexes in a truncated octaheꢀ
dron of water molecules obtained with the molecular mechanꢀ
CF2
O
NH(Lys)
I
O
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O
CF2
HO
ics force field AMBER was employed. The binding mode of
the intermediate I was also studied by manual replacement of
the ligand 1 with I in the binary 1@HSAꢀIA and 1@HSAꢀIIIA
complexes, which was then subjected to 60 ns of dynamic
simulation. Moreover, as for compound 1 the experimentally
observed stoichiometry ligand vs HSA was 2:1, the ternary
NH(Lys)
II
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In the case of 1, an increment of ca. 170 amu was
observed in peptides 99NECFLQHKDDNPNLPR114 (Mr exp.
2165.9548, Mr calc. 2165.9589) and
414KVPQVSTPTLVEVSR428 (Mr exp. = 1808.9629, Mr calc.
1808.9673). The MS/MS analysis revealed modification of
Lys106 (in subdomain IA). The MS/MS fragment ions showed
an unmodified y ion series from y to y , whereas an increment
of m/z 298 amu was detected at Lys106 between y and y9
1
+1@HSA complex was also considered. These studies reꢀ
=
vealed subꢀdomain IA as the main binding pocket of 1, which
will be discussed below.
=
(a) Binary 1@HSAꢀIA complex. The results from the dynamic
simulation of the 1@HSAꢀIA complex showed that the proꢀ
posed binding for 1 in subꢀdomain IA obtained by docking
was reliable as this complex proved to be very stable (Figure
2). Analysis of the rootꢀmeanꢀsquare deviation (rmsd) for the
2
8
8
corresponding to C H O –Lys(–H O). Besides, Lys414 (in
11
7
2
2
subꢀdomain IIIA) was also modified, with a Lys106:Lys 414
ratio of ca. 85. A suggested reaction mechanism is outlined in
Scheme 2. A similar analysis for 2 showed an increment of
96 amu in 191ASSAKQR197 (Mr exp = 942.4536, Mr calc. =
42.4559) with covalent binding at Lys195 (located in subꢀ
domain IIA). In this case, the MS/MS fragment ions showed
an unmodified y ion series from y to y , while an increment of
m/z 324 amu was detected at Lys195 between y and y correꢀ
sponding to C H O –Lys(–H O). Details of the ESIꢀMS/MS
spectra and fragmentation patterns are shown in Figures S10ꢀ
S13 in the SI.
whole protein backbone (C , C, N and O atoms) calculated in
α
the complex obtained from MD simulations studies revealed
that it varies between 1.0−3.0 Å (average of 1.6 Å) and is
relatively low for subꢀdomain IA (0.9−1.3 Å and an average of
1
9
0
.9 Å) (Figure S14). Ligand 1 would be anchored to subꢀ
3
6
domain IA of HSA via two hydrogen bonding interactions
involving the phenol group and the main carbonyl group of
Tyr148 and the amide side chain of Gln196 (Figures 2A and
2
3
13
9
2
2
2
B). Analysis of the variation of the relative distance between
the atoms involved in the aforementioned hydrogen bonding
interactions during the whole simulation revealed that 1 is well
fixed in this pocket and its conformation properly controlled
Scheme 2. Proposed covalent modification mechanism for 1.
by these residues (Figure 2C). The CF moiety would be also
3
located in the proximity of ɛꢀamino group of Lys106 with an
average distance of 5.3 Å during the simulation. In addition,
ligand 1 would stablish numerous apolar interactions within
the pocket, mainly involving the side chain of residues
Leu103, Ala151, Leu250, Gly248, Cys246, Cys200 and the
carbon side chain of Gln29.
(b) Binary I@HSAꢀIA complex. As it is shown in Figure 2D,
the results of our computational studies revealed that the inꢀ
termediate I would be anchored to subꢀdomain IA of HSA via
hydrogen bonding interaction with the main NH group of
Ala151. The analysis of the relative distance between the C19
atom (CF group) in I and the NZ atom of Lys106 and O17
2
Docking and Molecular Dynamics Simulations Studies. In
an effort to understand in atomic detail the covalent modificaꢀ
tion mechanism as well as to gain insight into the molecular
basis of the
atom (carbonyl group) in I and NH group of Ala151 during
the simulation revealed that this contact is present during 92%
of the simulation with an average distance of 3.0 Å (Figure
2
E). Furthermore, the difluoromethide moiety in I would be
observed selectivities, docking and Molecular Dynamics (MD)
simulation studies were carried out.
located closer to the ɛꢀamino group of Lys106 (average disꢀ
tance of 4.8 Å) than in the 1@HSAꢀIA complex (Figures 2C
vs 2E). This proposed arrangement of the intermediate I adeꢀ
quately explains the experimentally observed modification of
Lys106.
Binding of 1 to HSA and covalent addition to subꢀdomains IA
and IIIAꢀ The binding modes of ligand 1 in subꢀdomains IA
and IIIA of HSA were first studied by molecular docking
using the program GOLD version 5.2 and the available proꢀ
tein coordinates of the crystallographically determined HSA in
complex with iophenoxic acid (PDB code 2YDF). This
structure was chosen because two of the four molecules obꢀ
served are located close to Lys414. The positions of these
molecules in subꢀdomain IIIA and of the ɛꢀamino group of
25
Once the intermediate I is generated by irradiation of
the 1@HSA protein complex, covalent attachment would
probably occur by nucleophilic attack of Lys106, which would
be triggered by Asp108 acting as the general base and through
the generation of the tyrosinate form of Tyr148 (Figure 2D).
Thus, during 90% of the simulation a water molecule from the
26
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bulk solvent remained fixed between the side chain of Asp108
Asn391 (Figure 3B). Remarkably, the overall binding stability
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and Tyr148, with one of its protons engaged in a hydrogen
bond with a carboxylate oxygen of Asp108 and one of its
oxygen’s lone pairs accepting a hydrogen bond from the oxyꢀ
gen atom of Tyr148. Under this arrangement, the tyrosinate
could be generated and be the general base for the deprotonaꢀ
tion of Lys106 (nucleophile). The average distance between
the NZ atom of Lys106 and the O atom of Tyr148, the O
atom of Tyr148 and the O atom of the water molecule and
the closest OD1/OD2 atoms (carboxyl group) of Asp108
and the O atom of the water molecule during the whole
simulation is 5.50 Å, 2.85 Å and 2.73 Å, respectively (Figure
S15). The generation of catalytic tyrosinates in proteins by
neighbor aspartate residues, either involving or not a water
of ligand 1 in subꢀdomain IIIA seems to be lower than in subꢀ
domain IA in view of the greater mobility of the ligand in the
pocket. This is because the binding pocket of the subꢀdomain
IA is smaller and the polar interactions with the protein are
stronger. These findings also revealed that the primary binding
pocket of ligand 1 would be subꢀdomain IA. In this case, resiꢀ
due Glu492, which is located close by Lys414, would act as
the general base for the deprotonation of the lysine residue that
undergoes covalent modification. The average distance beꢀ
tween the NZ atom of Lys414 and the closest OE1/OE2 atoms
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(carboxylate grou) of Glu492 is 4.66 Å during the whole simuꢀ
lation (Figure S16).
(d) Ternary 1+1@HSA complex. The experimentally observed
2:1 complex was also studied (Figure 3D). In general, no
significant differences were observed in the binding of 1 to
subꢀdomain IA in the ternary and binary complexes, locating
28
molecule, has previously been reported.
(c) Binary 1@HSAꢀIIIA complex. In general, the computationꢀ
al studies carried out with the binary complex 1@HSAꢀIIIA
reveal that in contrast to the binary complex 1@HSAꢀIA in
in both cases the CF group of 1 in close contact to the ɛꢀ
3
amino group of Lys106 (average distance of 5.1 Å vs 5.3 Å,
respectively) (Figures 3E vs 2C, black lines). More importantꢀ
ly, the presence of 1 in this pocket would significantly reduce
which the CF group of the ligand is located close to the lysine
3
that is modified, Lys106, the latter group would be placed
further away from Lys414 (Figure 3). In particular, the averꢀ
age distance between the NZ atom of Lys414 and the C19
the distance between the ɛꢀamino group of Lys414 and the CF3
group in 1 for covalent modification. These findings also
atom (CF group) is of 9.0 Å during the 60 ns of simulation
3
reveal that: (1) subꢀdomain IA would be the primary binding
pocket of 1, and (2) this last binding would have a positive
cooperative effect that would favor the binding to subꢀdomain
IIIA.
(
Figure 3C, brown line). Moreover, the ligand would be locatꢀ
ed in this pocket due to a strong hydrogen bonding with the
main carbonyl group of Leu430 (average distance of 5.6 Å,
Figure 3C, yellow line) as well as diverse apolar interactions
with the side chain of residues Leu407, Val433, Leu453,
Ala449, Ile388, Leu387 and Phe403, and the carbon chain of
Figure 2. Proposed binding mode of 1 (yellow) and the intermediate I (green) in subꢀdomain IA as obtained by MD simulation
studies. (A) Overall view of the proposed binary 1@HSAꢀIA complex. Snapshot after 60 ns is shown. (B) Detailed view of the
1
@HSAꢀIA complex. (C) Variation of the relative distance between the C19 (CF group) and the O atom of 1 and the NZ atom of
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Lys106, the O atom of Tyr148 and the NE2 atom of Gln196, respectively, in the 1@HSAꢀIA protein complex during 60 ns of simuꢀ
lation. (D) Detailed view of the binary I@HSAꢀIA complex. Snapshot after 30 ns is shown. (E) Variation of the relative distance
between the C19 (CF group) in I and the NZ atom of Lys106 and O17 atom (carbonyl group) in I and NH group of Ala151 in the
2
I@HSAꢀIA protein complex during the whole simulation. Relevant side chain residues are shown and labelled. Lys106 and Asp108
are shown in orange and yellow, respectively. Hydrogen bonding interactions are shown as red dashed lines. Note how for both
complexes the lysine residue that is covalently modified (Lys106) is located very close to the fluoromethide moiety and the phenol
group in 1 and the ketone group in I would be anchored in the pocket by strong hydrogen bonding interactions.
0
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Figure 3. Proposed binding mode of 1 in binary 1@HSAꢀIIIA and ternary 1+1@HSA complexes as obtained by MD simulation
studies. (A) Overall view of the proposed binary 1@HSAꢀIIIA complex. Snapshot after 50 ns is shown. (B) Detailed view of the
1@HSAꢀIIIA complex. (C) Variation of the relative distance between the C19 (CF group) and O atom of 1 and the NZ atom of
3
Lys414 and the O atom of Leu430, respectively, in the 1@HSAꢀIIIA protein complex during 60 ns of simulation. (D) Overall view
of the proposed ternary 1+1@HSA complex. Snapshot after 20 ns is shown. (E) Variation of the relative distance between the C19
(
CF group) of 1 and the NZ atom of Lys106 and Lys414 in the 1@HSAꢀIIIA and 1+1@HSA complexes, respectively, during 60
3
ns of simulation. Relevant side chain residues are shown and labelled. Lys414 and Glu492 are shown in orange and yellow, respecꢀ
tively. Hydrogen bonding interactions are shown as red dashed lines. Note how Lys414 is located closer to the CF group in 1 in the
3
ternary 1+1@HSA complex than in the binary one.
The analysis of the rmsd for the whole protein backbone (Cα,
C, N and O atoms) and the subꢀdomains IA and IIIA calculatꢀ
ed in the ternary 1+1@HSA complex also corroborate the
increased stability of these protein pockets (Figure S9D). The
computational results explain the abovementioned photophysꢀ
ical and proteomic experimental results. Moreover, the MD
simulation studies carried out with the corresponding ternary
adducts revealed that the intermediate I would be rapidly
trapped, in particular by Lys414 as its mobility in this pocket
is very significant.
IIA of HSA was first studied by molecular docking using the
protein coordinates found in the crystal structure of HSA in
2
9
complex with oxyphenbutazone (PDB code 2BXB ). The
ligand in this structure is located close to the experimentally
observed modified Lys195 residue. It is important to highlight
that an analysis of the residues surrounding Lys195 revealed
that this residue is located nearby another lysine residue,
Lys199, whose modification was not observed (Figure 4).
Considering that when two lysine residues are located very
close in space, usually only one of them is protonated at physꢀ
iological pH, simulation studies with the two possible protonaꢀ
tion states of both lysine residues were performed. The best
Binding of 2 to HSA and covalent addition to subꢀdomain IIA
−
As for ligand 1, the binding mode of ligand 2 in subꢀdomain
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results were obtained considering neutral Lys195 (nucleoꢀ
ligand during the whole simulation rather than facing to the
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phile) and protonated Lys199, which would be also in agreeꢀ
ment with the experimental results. Furthermore, although the
obtained binding mode reveals that both residues are close in
distance to the CF3 group in 2, the arrangement of the side
chain of Lys199 observed during 60 ns of simulation revealed
to be geometrically inappropriate for nucleophilic attack once
the intermediate II would be generated (Figures 4B and 4D).
The ɛꢀamino group in Lys199 is predicted to be parallel to the
difluoromethide group in II for chemical modification. The
binary 2@HSAꢀIIA complex probed to be very stable, conꢀ
firming the reliability of the proposed binding (Figure S17).
Unlike ligand 1, direct polar contacts between ligand 2 and the
residues
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Figure 4. Proposed binding mode of the ligand 2 (violet) and the intermediate II (green) in subꢀdomain IIA as obtained by MD
simulation studies. (A) Overall view of the proposed binary 2@HSAꢀIIA complex. Snapshot after 60 ns is shown. (B) Detailed
view of the binary 2@HSAꢀIIA complex. (C) Variation of the relative distance between the C23 atom (CF group) and the NZ atom
of Lys195 in the II@HSAꢀIIA protein complex during 60 ns of simulation. (D) Detailed view of the II@HSAꢀIIA adduct. Snapshot
after 20 ns is shown. Note how Lys195 is well located for nucleophilic attack. Relevant side chain residues are shown and labelled.
Hydrogen bonding interactions of the intermediate II are shown as red dashed lines.
2
within the pocket were not identified. In this case, the polar
interactions of the phenol moiety in 2, which fixes this ligand
in the pocket, would be mediated by water molecules (not
shown). Moreover, ligand 2 would be embedded in a large
apolar pocket involving residues Val343, Val344, Val482,
Leu347, Leu481, Val455, Leu198, Phe206, Phe211, Ala210,
Trp214 and the carbon side chain of Asp451 and Ser202.
tween II and the protein were observed. Instead, the ketone
group in II would interact with the side chain of Ser202 and
Arg209 through water molecules. Under this arrangement, the
ɛꢀamino group of Lys195 is well located for nucleophilic
attack to the difluoromethide moiety in II. Thus, the analysis
of the variation of the relative distance between the C23 atom
(CF group) and the NZ atom of Lys195 in the II@HSAꢀIIA
2
protein adduct during 60 ns of simulation revealed an average
distance of 5.7 Å (Figure 4C).
The intermediate II generated by irradiation of the 2@HSAꢀ
IIA protein complex would have a similar binding mode as
ligand 2 (Figure 4D). As for 2, no direct polar contacts beꢀ
CONCLUSIONS
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2
The combined experimental and theoretical results suggest
that quinone methides photogenerated from trifluoroꢀ
methylphenols can be appropriate latent electrophiles to
achieve selective lysine targeting. Thus, 1 and 2 form groundꢀ
state complexes with HSA, characterized by a longꢀ
wavelength absorption band with maxima at ca. 320 nm,
which allows their selective excitation, resulting in a wellꢀ
defined emission appearing at ca. 420 nm. Photolysis of the
complexes produces a covalently modified protein, whose
proteomic analysis reveals selective targeting of Lys 106 and,
to a lesser extent, Lys 414, in the case of ligand 1; by contrast,
Lys 195 becomes selectively modified upon irradiation of
ligand 2 within HSA. The results of our MD simulation studꢀ
ies revealed that not only the ligands but also the photogenerꢀ
ated intermediates form stable complexes with the identified
pockets of HSA, which would explain the selective modificaꢀ
tion of the internal lysine residues, Lys106, Lys414, and
Lys195. The computational studies performed with the binary
different media, employing 10 × 10 mm quartz cells with 4
mL capacity.
Laser flash photolysis measurements. Experiments were
carried out with a pulsed Nd:YAG Laser system with 355 nm
as excitation wavelength, 0.6 cm as beam diameter and a pulse
duration of 10 ns. The energy was set at 15 mJ/pulse. The
apparatus consisted of the pulsed laser, a xenon lamp, a monoꢀ
chromator, and a photomultiplier. Samples of 1 and 2 in the
presence of 5 equivalents of HSA were placed in 10 mm × 10
mm quartz cells. The absorbance of the complexes at the exciꢀ
tation wavelength was kept at 0.25. Experiments were perꢀ
formed at 22°C.
0
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Steady-state photolysis experiments. Irradiations of 1 or 2 (5
10 M) were performed in PBS in the presence or absence
−5
×
of HSA (ligand/protein 1:5 molar ratio) in a multilamp photoꢀ
reactor containing low pressure mercury lamps (14 × 8 W),
with emission maximum at λ= 300 nm, through Pyrex. The
course of the process was followed by monitoring the changes
in the fluorescence spectra of the reaction mixtures at increasꢀ
ing times.
1
@HSAꢀIA and 1@HSAꢀIIIA and ternary 1+1@HSA comꢀ
plexes reveal subꢀdomain IA as the main binding pocket of 1.
Ligand 1 would be anchored to this pocket via two hydrogen
bonding interactions involving the phenol group and the main
carbonyl group of Tyr148 and the amide side chain of Gln19.
After irradiation, the photogenerated intermediate I would be
fixed to this pocket via hydrogen bonding interaction with the
main NH group of Ala151 allowing the difluoromethide moieꢀ
ty in I to be located close to the ɛꢀamino group of Lys106 for
nucleophilic attack. In addition, binding to subꢀdomain IA
would have a positive cooperative effect that would favor the
binding to subꢀdomain IIIA. This is in agreement with the
experimentally observed modification of Lys106 and Lys414
residues in a ratio of ca. 85:1, respectively. On the other hand,
the intermediate II photogenerated from ligand 2 would bind
to subꢀdomain IIA via polar contacts with the side chain of
Ser202 and Arg209 mediated by water molecules. Under this
arrangement, the ɛꢀamino group of Lys195 would be well
located for nucleophilic attack to the difluoromethide moiety
in II.
Protein Digestion and LC-ESI-MS/MS Analysis. Human
serum albumin (HSA) was enzymatically digested into smaller
peptides using trypsin. Subsequently, these peptides were
analyzed using nanoscale liquid chromatography coupled to
tandem mass spectrometry (nano LCꢀMS/MS). Briefly, 20 µg
of sample were taken (according to Qubit quantitation) and the
volume was set to 20 µL. Digestion was achieved with seꢀ
quencing grade trypsin (Promega) according to the following
steps: i) 2 mM DTT in 50 mM NH HCO V = 25 µL, 20 min
4
3
(
60 °C); ii) 5.5 mM IAM in 50 mM NH HCO V=30 µL, 30
4 3
min (dark); iii) 10 mM DTT in 50 mM NH HCO V = 60 µL,
0 min; iv) Trypsin (Trypsin: Protein ratio 1:20 w/w) V = 64
4
3
3
µL, overnight 37 °C. Digestion was stopped with 7 ꢁL 10%
TFA (Cf protein ca 0.28 µg/µL). Next, 5 ꢁL of sample (except
the main bands) were loaded onto a trap column (NanoLC
Column, 3ꢁ C18ꢀCL, 350 um × 0.5 mm; Eksigent) and desaltꢀ
ed with 0.1% TFA at 3 ꢁL/min during 5 min. The peptides
were then loaded onto an analytical column (LC Column, 3 ꢁ
C18ꢀCL, 75 um × 12 cm, Nikkyo) equilibrated in 5% acetoniꢀ
trile 0.1% formic acid. Elution was carried out with a linear
gradient of 5 to 45% B in A for 30 min (A: 0.1% formic acid;
B: acetonitrile, 0.1% formic acid) at a flow rate of 300
EXPERIMENTAL SECTION
General. Human serum albumin and 4’ꢀhydroxyꢀ4ꢀ
biphenylcarboxylic acid were commercially available. Specꢀ
trophotometric, HPLC or reagent grade solvents were used
without further purification. Buffered saline solutions at pH
ꢁ
L/min. Peptides were analyzed in a mass spectrometer
nanoESI qQTOF (5600 TripleTOF, ABSCIEX). The triꢀ
pleTOF was operated in informationꢀdependent acquisition
mode, in which a 0.25ꢀs TOF MS scan from 350–1250 m/z
was performed, followed by 0.05ꢀs product ion scans from
100ꢀ1500 m/z on the 50 most intense 2ꢀ5 charged ions. Proꢀ
teinPilot v4.5. (ABSciex) search engine default parameters
were used to generate peak list directly from 5600 TripleTOF
wiff files. The obtained mgf was used for identification with
MASCOT (v 4.0, Matrixꢀ Science). Database search was
performed on SwissProt database. Searches were done with
tryptic specificity allowing one missed cleavage and a tolerꢀ
ance on the mass measurement of 100 ppm in MS mode and
7
.4 and pH 12 were prepared by dissolving commercially
available tablets in deionized water. Ligands 4ꢀ
trifluoromethylꢀ1ꢀnaphthol (1) and 4ꢀ(4ꢀ
trifluorometylphenyl)phenol (2), were synthesized following
3
0
previously reported procedures.
Absorption and emission measurements. Optical spectra at
different media were recorded on a JASCO Vꢀ630 spectrophoꢀ
tometer. Steadyꢀstate emission and excitation spectra were
recorded on a JASCO FPꢀ8500 spectrofluorometer system,
provided with a monochromator in the wavelength range of
200ꢀ850 nm, at 22°C. Timeꢀresolved fluorescence measureꢀ
ments were performed with an EasyLife V spectrometer from
0
.6 Da in MS/MS mode.
OBB (Palaiseau, France), equipped with a pulsed LED (λexc
Docking studies. They were carried out using program GOLD
.2.2 and the protein coordinates found in the crystal strucꢀ
ture of HSA in complex with: (a) oxyphenbutazone (PDB
code 2BXB ) for 2 and iophenoxic acid (PDB code 2YDF )
for 1. These structures were selected since the observed ligꢀ
2
95 nm) as excitation source. The kinetic traces were fitted by
23
5
one monoexponential decay function, using a deconvolution
procedure to separate them from the lamp pulse profile. Experꢀ
iments were performed on solutions of known concentration in
29
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ands are located in the same region where the lysine residues
ent); (b) minimization of the ligand (1000 steps, first half
2
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8
9
1
1
1
1
1
1
1
1
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1
2
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covalently modified by 1 and 2 were observed. Ligand geomeꢀ
using steepest descent and the rest using conjugate gradient);
(c) minimization of the solvent and ions (5000 steps, first half
using steepest descent and the rest using conjugate gradient);
(d) minimization of the side chain residues, waters and ions
(5000 steps, first half using steepest descent and the rest using
conjugate gradient); (e) final minimization of the whole sysꢀ
tem (5000 steps, first half using steepest descent and the rest
using conjugate gradient). A positional restraint force of 50
tries were minimized using the AM1 Hamiltonian as impleꢀ
3
1
mented in the program Gaussian 09 and used as MOL2 files.
Each ligand was docked in 25 independent genetic algorithm
(GA) runs, and for each of these a maximum number of
1
00000 GA operations were performed on a single population
of 50 individuals. Operator weights for crossover, mutation
and migration in the entry box were used as default parameters
–1
–2
(
(
95, 95, and 10, respectively), as well as the hydrogen bonding
4.0 Å) and van der Waals (2.5 Å) parameters. The position of
kcal mol
Å
was applied to not minimized residues of the
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protein during the stages aꢀc and to α carbons during the stage
d, respectively.
the side chain of the experimentally observed modified residue
was used to define the activeꢀsite and the radius was set to 8
Å. All crystallographic water molecules and the ligands were
removed for docking. The “flip ring corners” flag was
switched on, while all the other flags were off. The GOLD
scoring function was used to rank the ligands in order to fitꢀ
ness.
(
c) Simulations. MD simulations were performed using the
38ꢀ40
pmemd.cuda_SPFP
module from the AMBER 14 suite of
programs. Periodic boundary conditions were applied and
electrostatic interactions were treated using the smooth partiꢀ
41
cle mesh Ewald method (PME) with a grid spacing of 1 Å.
The cutoff distance for the nonꢀbonded interactions was 9 Å.
42
Molecular dynamic simulations. (a) Ligand minimization.
The ligand geometries of the highest score solution obtained
by docking were minimized using a restricted Hartree–Fock
The SHAKE algorithm was applied to all bonds containing
−5
hydrogen using a tolerance of 10 Å and an integration step of
2.0 fs. The minimized system was then heated at 300 K at 1
atm by increasing the temperature from 0 K to 300 K over 100
ps and by keeping the system at 300 K another 100 ps. A
(RHF) method and a 6–31G(d) basis set, as implemented in
the ab initio program Gaussian 09. Partial charges were deꢀ
rived by quantum mechanical calculations using Gaussian 09,
–1
–2
positional restraint force of 50 kcal mol
Å
was applied to
32
as implemented in the R.E.D. Server (version 3.0), according
all α carbons during the heating stage. Finally, an equilibration
33
to the RESP model. Ligand coordinates obtained by docking
of the system at constant volume (200 ps with positional reꢀ
–1
–2
were employed as starting point for MD simulations. The
missing bonded and nonꢀbonded parameters were assigned, by
analogy or through interpolation, from those already present in
straints of 5 kcal mol
Å
to α alpha carbons) and constant
pressure (another 100 ps with positional restraints of 5 kcal
Å to α alpha carbons) were performed. The positional
restraints were gradually reduced from 5 to 1 mol
–1
–2
mol
34
35
the AMBER database (GAFF ).
–1 –2
Å
(5
(b) Generation and minimization of the complexes. Simulaꢀ
tions of the HSA/2 and HSA/1 binary complexes were carried
out using the enzyme geometries in PDB codes 2BXB and
steps, 100 ps each), and the resulting systems were allowed to
equilibrate further (100 ps). Unrestrained MD simulations
were carried out for 100 ns. System coordinates were collected
every 10 ps for further analysis. The molecular graphics proꢀ
2
YDF, respectively. Computation of the protonation state of
++
43
titratable groups at pH 7.0 was carried out using the H Web
server. Addition of hydrogen and molecular mechanics paꢀ
rameters from the ff14SB and GAFF force fields, respectiveꢀ
ly, were assigned to the protein and the ligands using the LEaP
module of AMBER Tools 15. As a result of this analysis: (i)
gram PyMOL was employed for visualization and depicting
36
ligand/protein structures. The cpptraj module in AMBER 14
was used to analyse the trajectories and to calculate the rootꢀ
meanꢀsquare deviations (rmsd) of the protein during the simuꢀ
3
7
29
44
lation.
His535 was protonated in δ position; (ii) His3, His9, His39,
His146, His242, His288, His440, His464 and His510 were
protonated in ε position; (iii) His67, His105, His128, His247,
His338 and His367 were protonated in δ and ε positions. The
protein was immersed in a truncated octahedron of ∼25000
TIP3P water molecules and neutralized by addition of sodium
ions. The system was minimized in five stages: (a) minimizaꢀ
tion of poorly unsolved residues: (i) for PDB 2BXB: 12, 33,
ASSOCIATED CONTENT
Supporting Information: Experimental procedures for the synꢀ
thesis and characterization of compound 1−2, docking and MD
simulation studies. Extra figures of the phothophysical studies
(Figures S1ꢀS9), proteomic analysis (Figures S10ꢀS13), MD
simulation studies (Figures S14−S17). "The cartesian coordinates
of all the complexes included in this work are also provided." This
material is available free of charge via the Internet at
http://pubs.acs.org.
4
1
3
5
5
5
1
1
1, 51, 56, 60, 73, 81, 82, 84, 94, 95, 97, 111, 114, 174, 186,
90, 205, 209, 225, 227, 240, 250, 275, 276, 277, 297, 301,
13, 317, 321, 359, 390, 402, 436, 439, 444, 466, 513, 519,
24, 532, 536, 538, 541, 545, 550, 551, 560, 562, 564, 565,
74 and 580; and (ii) for PDB 2YDF: 12, 16, 31, 33, 41, 48,
6, 60, 64, 69, 77, 81, 82, 84, 93, 94, 95, 97, 98, 104, 109,
14, 115, 119, 121, 132, 136, 137, 141, 159, 160, 162, 167,
74, 178, 186, 190, 195, 205, 262, 269, 276, 277, 280, 301,
AUTHOR INFORMATION
Corresponding Authors
*mcjimene@qim.upv.es;
*mmiranda@qim.upv.es;
*concepcion.gonzalez.bello@usc.es
302, 305, 313, 314, 317, 351, 359, 365, 372, 376, 389, 390,
397, 402, 425 ,432, 436, 439, 444, 459, 466, 500, 502, 503,
505, 507, 509, 510, 512, 513, 516, 520, 524, 525, 536, 541,
ORCID
Raúl PérezꢀRuiz: 0000ꢀ0003ꢀ1136ꢀ3598
5
5
43, 545, 548, 549, 550, 551, 554, 560, 562, 563, 564, 565,
68, 570, 571, 573, 574, 575, 580 and 582 (1000 steps, first
Emilio Lence: 0000ꢀ0001ꢀ9489ꢀ9421
Concepción GonzálezꢀBello: 0000ꢀ0001ꢀ6439ꢀ553X
half using steepest descent and the rest using conjugate gradiꢀ
ACS Paragon Plus Environment

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Miguel A. Miranda: 0000ꢀ0002ꢀ7717ꢀ8750
M. Consuelo Jiménez: 0000ꢀ0002ꢀ8057ꢀ4316
Cravatt, B. F.; Forli, S.; Powers, E. T.; Sharpless, K. B.; Wilson, I.
A.; Kelly, J. W. "Inverse Drug Discovery" Strategy to Identify
Proteins that are Targeted by Latent Electrophiles as Exemplified
by Aryl Fluorosulfates. J. Am. Chem. Soc. 2018, 140, 200−210.
(6) (a) Freccero, M. Quinone Methides as Alkylating and Crossꢀ
Linking Agents. MiniꢀReviews Org. Chem. 2004, 1, 403–415. (b)
Wang, P.; Song, Y.; Zhang, L.; He, H.; Zhou, X. Quinone Methide
Derivatives: Important Intermediates to DNA Alkylating and DNA
CrossꢀLinking Actions. Curr. Med. Chem. 2005, 12, 2893–2913. (c)
Van De Water, R. W.; Pettus, T. R. R. OꢀQuinones Methides: Inꢀ
termediates Underdeveloped and Underutilized in Organic Syntheꢀ
sis. Tetrahedron 2002, 58, 5367–5405. (d) McCracken, P. G.; Bolꢀ
ton, J. L.; Thatcher, G. R. J. Covalent Modification of Proteins and
Peptides by the Quinone Methide from 2ꢀtertꢀButylꢀ4,6ꢀ
dimethylphenol:ꢂ Selectivity and Reactivity with Respect to Comꢀ
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J. L.; Turnipseed, S. B.; Thompson, J. A. Chem. Biol. Interact.
ACKNOWLEDGMENTS
Financial support from the Spanish Ministry of Economy and
Competiveness [CTQ2016ꢀ78875ꢀP, SAF2016ꢀ75638ꢀR and
BES‐ 2014‐ 069404 (preꢀdoctoral fellowship to O. M.ꢀM.)], the
Generalitat Valenciana (PROMETEO/2017/075), the Community
of Madrid (2016ꢀT1/AMBꢀ1275), the Xunta de Galicia (Centro
Singular de Investigación de Galicia accreditation 2016−2019,
ED431G/09 and postꢀdoctoral fellowship to E. L.) and the Euroꢀ
pean Union (European Regional Development Fund − ERDF) is
gratefully acknowledged. The proteomic analysis was performed
in the proteomics facility of SCSIE University of Valencia that
belongs to ProteoRed PRB2ꢀISCIII and is supported by grant
PT13/0001, of the PE I+D+i 2013–2016, funded by ISCIII and
FEDER.
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