Supporting Information - Royal Society of Chemistry

Transcrição

Electronic Supplementary Material (ESI) for Dalton Transactions
This journal is © The Royal Society of Chemistry 2013
Supporting Information
“Screening organometallic binuclear thiosemicarbazone ruthenium complexes as potential
anti-tumour agents: cytotoxic activity and human serum albumin binding mechanism”
Bruno Demoro,a Rodrigo F. M. de Almeida,b Fernanda Marques,c Cristina P. Matos,d Lucía Otero,a João Costa
Pessoa,e Isabel Santos,c Alejandra Rodríguez,f Virtudes Moreno,f Julia Lorenzo,g Dinorah Gambinoa and Ana
Isabel Tomaz*d
a
Cátedra de Química Inorgánica, Facultad de Química, Universidad de la República, 11800 Montevideo, Uruguay
b
Centro de Química e Bioquímica, Faculdade de Ciências da Universidade de Lisboa, 1749-016 Lisbon, Portugal;
c
Unidade de Ciências Químicas e Radiofarmacêuticas, Instituto Superior Técnico, Pólo de Loures - Campus
Tecnológico e Nuclear, Estrada Nacional 10, 2686-953 Sacavém, Portugal;
d
Centro de Ciências Moleculares e Materiais, Departamento de Química e Bioquímica, Faculdade de Ciências da
Universidade de Lisboa, Campo Grande, 1749-016 Lisbon, Portugal ([email protected])
e
Centro de Química Estrutural, Instituto Superior Técnico, Universidade Técnica de Lisboa, Av. Rovisco Pais, 1049-001
Lisbon, Portugal
f
Departament de Química Inorgànica, Universitat de Barcelona, Martí i Franquès 1-11, 08028 Barcelona, Spain
g
Institut de Biotecnologia i Biomedicina, Universitat Autònoma de Barcelona, 08193-Bellaterra, Barcelona, Spain
Supporting Information includes the following Sections:
1. Cell Studies
with figures
SI.1 – In vitro cytotoxic activity of ligands L1-L4 against A2780, MCF7 and PC3 cell lines;
SI.2 – In vitro cytotoxic activity of the thiosemicarbazone (TSC) ligands;
SI.3 – Dose-response profiles of cell viability in A2780, MCF7 and PC3 cell lines;
2. Time-dependent induced CD spectra for HSA binding of complexes 1 and 4
with explaining text, and figures
SI.4 – Binding of complex 1 to HSA monitored by CD spectroscopy (time dependence and equilibrium conditions);
SI.5 – Time dependence of binding of complex 4 to HSA;
SI.6 – UV-Visible spectra on time dependence of complex 4 binding to HSA.
3. Mechanism of Förster Resonance Energy Transfer (FRET) for the HSA–complex 4 system
SI.7 – Spectral overlap of the HSA fluorescence emission and complex 4 absorbtion.
SI-1/9
1. Cell Studies
A total of 9 compounds – which comprise TSC ligands L1 to L4, the [Ru2(p-cym)2Cl4] precursor and
[Ru2(p-cym)2(TSC)]X2 complexes 1 to 4 (see Scheme 1) – were screened for their cytotoxic activity
against human tumour cell lines and compared against cisplatin (CDDP).
Figure SI.1. In vitro cytotoxic activity of all thiosemicarbazone compounds 1-4 and L1-L4 against the human
tumour cell lines A2780 (ovarian adenocarcinona), MCF-7 (breast adenocarcinoma) and PC-3 (grade IV prostate
carcinoma). IC50 values are reported in µM for a 72h incubation period. CDDP is cisplatin incubated in the same
conditions (IC50 values from50 for MCF7 and PC3 cells obtained with the same experimental methodology).
SI-2/9
TSC ligands L1, L2 and L3 are much more active than cisplatin against the MCF7 cell line, and exhibit
IC50 values close to those of CDDP in the very aggressive PC3 cell line as well (Figure SI.2.). The
cytotoxicity of complexes 1 and 3 is comparable to that of CDDP in MCF7 and in PC3 cells. While L4 is
typically not cytotoxic, complex 4 is in contrast quite active against all tumorigenic cell lines tested.
Figure SI.2. In vitro cytotoxic activity of the thiosemicarbazone (TSC) ligands L1-L4 against A2780, MCF7 and
PC3 cells. Cisplatin (CDDP) was included for comparison using the same experimental conditions (CDDP data
from 47 for MCF7 and PC3 cell lines). IC50 values are reported in µM for a 72h incubation period.
SI-3/9
L1
L2
L3
L4
RuL1
RuL2
RuL3
RuL4
A2780
A2780
150
% cellular viability
% cellular viability
150
100
50
0
-10
-8
-6
-4
100
50
0
-10
-2
-8
-3
-6
-4
-2
log C (mol/dm-3)
log C (mol/dm )
RuL1
RuL2
RuL3
RuL4
L1
L2
L3
L4
MCF7
MCF7
150
% viability
% viability
150
100
50
0
-10
-8
-6
-4
100
50
0
-10
-2
-8
-6
log C (mol/dm )
PC3
100
L1
80
L2
60
L3
L4
40
20
-8
-6
log C (mol/dm -3)
-4
-2
Celular viability
(% of control)
Celular viability
(% of control)
-2
PC3
100
0
-10
-4
log C (mol/dm-3)
-3
RuL1
80
RuL2
60
RuL3
RuL4
40
20
0
-10
-8
-6
-4
-2
log C (mol/dm -3)
Figure SI.3. Dose-response profiles of cell viability in the human tumor cell lines obtained for all TSC ligands L1L4 and their corresponding complexes 1-4 after a 72 h incubation period (complex concentrations are indicated in
M (mol.dm-3). IC50 values obtained are reported in Table 1.
SI-4/9
2. Time-dependent induced CD spectra for HSA binding of complexes 1 and 4
Time dependence for HSA binding of complexes 1 and 4 was monitored by CD spectroscopy since both
complexes yield an induced CD signal upon binding to the protein.
Figure SI.4 shows a weak ICD signal (consisting of two negative bands at ~ 400 nm (396 nm, band 1)
and ~500 nm (494 nm, band 2) detected after 1h and due to the binding of 1 to HSA. The fact that an
ICD signal is observed after a short incubation time suggests that the complex does neither dissociates
into monomeric species nor loses any of the coordinated ligands, and the binding of 1 to HSA is most
likely to involve the original dimeric complex. This is supported by the UV-Visible spectrum of complex
1 after 1 h incubation with HSA, that shows in the lower energy absorption band a ~25 nm red shift from
415 nm (in the free complex) to 440 nm (in the HSA-bound form); further, the UV-Visible spectrum of 1
in the presence of the protein (for λ>350 nm) does not change during 24 h of contact time – data not
shown.
The relative intensity of the two ICD bands 1 and 2 change with increasing incubation time (Figure
SI.4.A and SI.4.B), with a concomitant blue shift (ca. 10 nm) in Δεmax for both. In equilibrium conditions
the same two ICD bands are observed and their relative intensity seems to be concentration dependent
(Figure SI.4.C): while band 1 is intensified by increasing the complex concentration, only modest
changes are observed for band 2 after the 1:1 {complex 1: protein} molar ratio. These observations can
be tentatively explained supposing the existence of two different binding sites for complex 1 in HSA.
One of the sites probably becomes saturated at a 1:1 molar ratio in equilibrium conditions. The time
dependence of the relative intensity of the two bands suggests a kinetic preference for one of the binding
sites (possibly more solvent accessible), yielding in due time a more stable thermodynamic interaction.
Upon addition of a second mole equivalent of complex (Figure 4.C), the pattern of the CD signal
changes: band 1 (at ca. 385 nm) increases intensity, while band 2 (at ca. 500 nm) shifts to the red.
Therefore, it is clear that complex 1 binds to HSA, this binding occurring quite fast. If an excess of
complex is added (not probable in vivo), a second weaker binding site (corresponding to a distinct CD
signal) is also available.
This hypothesis is corroborated by the fluorescence measurements (see section 4.4.2). Steady-state and
time resolved fluorescence data for the binding of 1 to HSA is consistent with two different interactions:
one involving the loose binding of 1 to the protein (accounting for a dynamic quenching process
observed) and a binding site within the van der Waals distance of Trp214 (originating the static
quenching process).
SI-5/9
A
0.4
B
0.5
C
1
-0.2
1h
-0.4
2h
-0.6
3h
-0.8
4h
0.5
0
0
-0.5
-1
1h
3h
4h
22h
-1.5
6h
-1
320
360
400
440
480
520
560
600
λ/nm
320
360
400
440
480
520
0
0.5:1 (**)
1:1 (22h)
2:1 (22h)
-3
560
600
2:1 (22h)*
320
360
400
-0.6
-0.7
-0.8
-0.6
-0.9
-1.2
6
8
10
480
520
560
600
640
680
494 nm
**
-0.5
-1
-1.5
-2
-1.5
incubation time (h)
440
396 nm
0
396 nm
494 nm
Δε / (M-1 cm-1)
Δε / (M-1 cm-1)
-0.5
4
-2
-2.5
λ/nm
-0.3
-0.4
2
-1
-1.5
-3.5
396 nm
494 nm
-0.3
0
-0.5
λ/nm
-0.2
Δε / (M-1 cm-1)
-2
Δε / (M-1 cm-1)
0
Δε / (M-1 cm-1)
Δε / (M-1 cm-1)
0.2
0
5
10
15
incubation time (h)
20
25
-2.5
0
0.5
1
1.5
2
2.5
molar ratio [complex 1]:[HSA]
3
Figure SI.4. Binding of complex 1 to HSA monitored by CD spectroscopy: A) Time dependence: top - Induced CD
(ICD) signal recorded for a solution of HSA (100 µM) with 1 at a 1:0.5 protein:complex molar ratio incubated with
time at 37ºC (arrows indicate changes observed with increasing incubation time); bottom - corresponding evolution
of the molar differential absorption values (Δε) for ICD band 1 (circles) and band 2 (triangles) with increasing
incubation time; B) Time dependence till equilibrium conditions: top - ICD signal recorded for a solution of HSA
(100 µM) with 1 at a 1:1 protein:complex molar ratio incubated with increasing time up to 22 h at 37ºC; bottom corresponding evolution of Δε for band 1 (circles) and band 2 (triangles) with increasing incubation time; C) ICD
spectra in equilibrium conditions. top - Changes in ICD signal with increasing concentration of 1 (*: noise-filtered
spectrum for the 1:2 protein:complex molar ratio; **: ICD spectrum for 6 h incubation time): bottom - Changes in
Δε observed for band 1 (circles) and band 2 (triangles) with increasing concentration of complex 1.
ICD spectra observed in solutions containing HSA and 4 are far more intense than those recorded for 1,
exhibiting a quite different pattern that also changes with increasing incubation time – Figure SI.5. Strong
ICD signals are recorded after as short as a 10 min incubation time (Figure SI.5.A1) showing several bands
– an intense positive one at ~500 nm (503 nm), a less intense negative band at ~ 400 nm (416 nm) and a third
negative band at ca. 340 nm (possibly another one hinted at lower wavelengths, also negative). With
increasing incubation time the intensity of all ICD bands slightly increases with a concomitant blue shift of
ca. 10 nm up to 1 h, after which the intensity of the positive ICD starts decreasing (Figure SI.5.A2 and
SI.5.A3). In equilibrium conditions (after 24 h) a completely different CD spectrum is observed with
inversion of the signal of the two main ICD bands of lower energy (Figure SI.5.B).
SI-6/9
10
8
8
6
6
4
4
∆ε /(M-1 cm-1)
∆ε /(M-1 cm-1)
A2
A1
10
2
0
-2
60 min
10 min
-4
20 min
40 min
-6
HSA alone
-2
HSA alone
-4
2
0
80 min
100 min
-6
60 min
120 min
-8
-8
300
400
500
600
300
400
500
600
λ / nm
λ / nm
11
A3
500 nm
10
1h
8
9
B
HSA alone
0
410 nm
3h
6
5
-3
3
-4
1
∆ε /(M-1 cm-1)
-2
∆ε ( •) / (M-1Cm-1)
∆ε (▲) / (M-1Cm-1)
-1
7
4h
22h
4
2
0
-2
-4
-6
-1
-5
0
50
100
time / min
150
200
-8
320
360
400
440
480
λ / nm
520
560
600
Figure SI.5. Time dependence of binding of complex 4 to HSA: A1/A2) Induced CD (ICD) signal for a 100 µM
solution of HSA incubated with 4 at a 1:2 protein:complex molar ratio recorded at (32 ± 2) ºC up to 2h, and A3)
corresponding evolution of the molar diferencial absorption values (Δε) for the positive 500 nm (triangles, left Y
axis) and the negative 410 nm (circles, right Y axis) ICD bands with increasing incubation time; B) Evolution of the
ICD signal for higher incubation times highlighting the inversion of the Cotton effect in equilibrium conditions
(signal recorded at room temparature for a 100 µM HSA solution incubated with 4 at a constant 1:1 molar ratio
kept with stirring at (37±0.5) ºC between measurements). Arrows highlight the changes observed with increasing
time in all ICD spectra.
The strong ICD signal observed after an incubation time as short as 10 min suggests that the complex is
binding to the protein as a whole entity. This is also supported by the UV-Visible spectra of 4 in the absence
and in the presence of the protein (Figure SI.6). The low energy band shows a ~30 nm red shift after 10 min
(at 32±2 ºC), followed by a small blue shift and an intensity loss with increasing incubation time up to 1h (~3
nm), after which the absorbance increases slightly until 2 h (Figure SI-6.C). After 22 h incubation, the
spectrum of HSA-bound complex 4 shows λmax at 455 nm (25 nm red shift) and a 23% hypocromism when
compared to that of the free complex (see main text, section 4.4.1, for the conclusions drawn).
SI-7/9
Figure SI.6. Time dependence of HSA binding for complex 4: A+B) UV-Visible spectra recorded for complex 4 (200 µM, dashed black line) and (colored full lines)
for a solution of HSA:4 at a 1:2 molar ratio (100 µM : 200 µM) recorded over time up to 2h at; C) Change in λmax (blue closed circles, •) and in the corresponding
absorbance (red full triangles,▲) in the spectra of 4 upon HSA binding over time (up to 2h). Samples in pH7.4 PBS/1%DMSO incubated at T = (32±2) º C; CD
spectra were recorded at the same temperature.
SI-8/9
3. Mechanism of Förster Resonance Energy Transfer (FRET) on the HSA–complex 4 system
Figure SI.7. Spectral overlap in 2% DMSO/PBS of the HSA fluorescence emission and complex 4
absorption: (red dashed line, ---) Absorbance spectrum obtained for a 200 µM solution of complex 4 (0.5
cm path quartz cuvette); (blue full line, ––) Trp214 emission of HSA (λexc=295 nm).
SI-9/9

Supporting Information - Royal Society of Chemistry

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