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F. Wang et al. / European Journal of Medicinal Chemistry 101 (2015) 126e132
substituted indolin-2-ones in the presence of triethylamine to form
target compounds 1e19.
still not well understood at present, due to the lack of a high res-
olution crystal structure for human P-gp. Molecular docking was
performed to understand the interaction between 4-thiazolidinone
derivatives containing indolin-2-one moiety and P-gp, which
would provide useful information for the further optimization of
those derivatives. The results indicate that there is no significant
difference amongst the binding energies between P-gp and de-
rivatives from each category (data not shown). However, Ser222, a
residue in TM4 domain of P-gp [19], exhibits an intriguing feature
in that it interacts with all of the derivatives related with P-gp
transport in a significant way, including both typical substrates and
modulators of P-gp. Furthermore, Ser222 even interacts with all of
the weak substrates of P-gp, except for the 2Z, 5Z isomer of com-
pound 19. Meanwhile, the compounds showing no interaction with
Ser222 are mainly from the category of non-substrates of P-gp
(only the 2Z, 5Z isomer of compound 19 is from the category of
weak substrates of P-gp). Ser222 also could interact with rhoda-
mine 123 and verapamil, the control compounds for substrate and
modulator of P-gp, respectively, at preferred ligand docking clus-
ters. Furthermore, the results of molecular docking also showed
that most of the P-gp substrate and modulator derivatives could
interact with Pro223, the neighbor residue of Ser222 within P-gp
(data not show). This could mean that Pro223 may act as an as-
sistant for Ser222 and could increase the interaction efficacy be-
tween P-gp and the derivatives, regardless of substrates or
modulators. The representative results could be seen in Fig. 1 and
Fig. 2.
The homology modeled human P-gp structure represents the
initial stage of the transport cycle of P-gp [3]. Furthermore, Ser222
locates at the entrance gate to the drug binding cavity of P-gp and is
involved in the interaction between P-gp and its substrates [19].
The research of Loo and Clark also has confirmed that Ser222 could
bind with verapamil, a representative modulators of P-gp [20].
Therefore, we proposed that Ser222 should act as one of the trig-
gers for the initiation of the transport cycle of P-gp, which means
that the binding of Ser222 with substrate will activate ATPase ac-
tivity of P-gp and cause P-gp to undergo large structural changes to
transport substrate outside of the cell. The initiation of P-gp
transport should be independent with the following substrate
translocation during the whole transport cycle. The modulators
inhibit the transport activity of P-gp towards substrate by blocking
the binding between the trigger residues within P-gp and substrate.
However, they could not be transported by P-gp as they could not
bind with the residues responsible for substrates translocation.
Therefore, the molecular docking study based on the initial stage
structure of P-gp only could be used to distinguish non-substrates
of P-gp from those that could interact with P-gp, including both
substrates and modulators of P-gp.
3. Results and discussion
3.1. Biological evaluation and molecular docking study on the
interaction of 4-thiazolidinone derivatives containing indolin-2-one
moiety (as mixtures of both isomers) with P-glycoprotein
The transport activity of P-gp to 4-thiazolidinone derivatives
containing indolin-2-one moiety and the transport inhibition ac-
tivities of the derivatives to P-gp were evaluated by cell (K562/S
and K562/ADM) based cytotoxicity assay and P-gp mediated
rhodamine 123 efflux inhibition assay, respectively. IC50 values of
the derivative against K562, the IC50 ratio between the IC50 values
of the tested derivative against K562/ADM and K562/S and the Ki
values of the derivatives against P-gp mediated rhodamine 123
efflux are shown in Table 1. The values of IC50 are the mean of at
least three independently performed experiments. Generally,
inter-experimental variation was below 20%. The control of adria-
mycin, a representative substrate of P-gp, shows an IC50 ratio of
6.75 (data not shown). AutoDock 4 and homology modeled human
P-gp structure [18] were then used to study the interaction be-
tween P-gp and the derivatives. Meanwhile, rhodamine 123 and
verapamil, the representative substrate and modulator of P-gp, are
used as control compounds for the study on the interaction be-
tween P-gp and its substrates and modulators. The interaction
between the derivatives and Ser222 within P-gp is shown in
Table 1.
As Table 1 shows, 4-thiazolidinone derivatives containing
indolin-2-one moiety exhibit multi-interaction types with P-gp,
even with similar molecular structures. Based on the data of IC50
ratio and Ki, the tested derivatives could be classified as typical
substrates of P-gp (The compounds show IC50 ratio values more
than 2 and Ki less than 2.23
the head note of “a”), typical modulators of P-gp (The compounds
show IC50 ratio values less than 1.3 and Ki less than 2.23 M. The
mM. The compounds were labelled with
m
compounds were labelled with the head note of “b”) and typical
non-substrates of P-gp (The compounds show IC50 ratio values less
than 1.3 and Ki more than 2.23 mM. The compounds were labelled
with the head note of “c”) [23]. Meanwhile, compounds 2, 5, 6, 14
and 19 show IC50 ratio values ranging from 1.32 to 1.98 and Ki more
than 2.23 mM. Therefore, those compounds were classified as weak
substrates of P-gp. Compounds 1, 2, 3, 4, 5, 6, 11, 14, 15, 16 and 19
are of substrates of P-gp, which means those compounds could not
overcome the MDR mediated by P-gp. Therefore, those compounds
show no value for further development. Compounds 7, 10, 12 and
13 are of tumor cell cytotoxic P-gp modulators, which show IC50
ratios ranging from 0.48 to 1.20 and Ki ranging from 0.12
m
M to
Accurate computational prediction of P-gp protein structure
represents a good starting point for molecular docking study. The
annotation of the function of Ser222 in P-gp has proven a successful
homology model constructed by G.E. Jara. The issue of substrate
translocation within P-gp should be addressed by the combination
of biochemical experiment, molecular docking and molecular dy-
namics simulations.
1.78 M. Many anti-tumor drugs in clinical use are being chal-
m
lenged by MDR caused by multi-mechanisms [24]. P-gp mediated
MDR is one of the major obstacles for tumor clinical chemotherapy.
Therefore, a P-gp modulator anti-tumor drug will increase the ef-
ficacy of another anti-tumor drug with the property of P-gp sub-
strate when they are administrated together to tumor patients.
Thus, the four derivatives show a more promising future than the
others at this point. Furthermore, there are still a few of the
compounds which can not be transported by P-gp, nor can they
inhibit P-gp mediated rhodamine 123 transport, such as com-
pounds 8, 9, 17 and 18. Those derivatives that are of non-substrates
of P-gp, also demonstrate a promising future for further separation
and development.
4. Conclusions
In summary, 4-thiazolidinone derivatives containing indolin-2-
one moiety exhibit multi-interaction types with P-gp, which would
provide crucial information for the further separation and devel-
opment on those derivatives. The further separation and develop-
ment should be focused on compounds 7, 10, 12 and 13 (tumor cell
cytotoxic P-gp modulators) and compounds 8, 9, 17 and 18 (non-
substrates of P-gp). Ser222 may be acting as one of the triggers
Understanding the interaction mechanism between anti-tumor
compounds and P-gp is crucial for the further optimization of these
compounds. The molecular mechanism of drug transport by P-gp is