Classes of tested compounds.
All compounds depicted in Fig. 1 were prepared following recently reported procedures15. The synthesis of 2-oxoquinoline-3-carbaldehydes (Ia-c) (Supplementary Fig. S1, online) started with the building of the quinoline core via the Meth-Cohn method15,16 from the corresponding acetanilides by treatment under Vielsmeier’s conditions, which gave rise to 2-chloroquinoline-3-carbaldehyde derivatives. These were transformed into the corresponding quinolone derivatives (Ia-c) by aqueous acetic acid hydrolysis, and were then reduced with sodium borohydride to 3-(hydroxymethyl)quinolinones (IIa-c), which with thionyl chloride afforded the 3-(chloromethyl)quinolinones (IIIa-c) (Supplementary Fig. S1, online). The starting material 2-chloro-4-(4-aryl)pyrimidines (1a-b) (a for 4-chlorophenyl and b for naphtha-2-yl) were prepared by Suzuki reaction from commercial 2,4-dichloropyrimidine with the corresponding arylboronic acid17,18 and used as precursors to give the pyrimidine derivatives 2–11 by aromatic nucleophilic substitution (see Supplementary Fig. S2, for reaction conditions, online): the amino residue linked to the pyrimidine ring at 2 position being, respectively, p-aminoacetophenone (2, 3),
p-phenylenediamine (4, 5), piperazine (6, 7), m-aminophenol (8, 8a), p-aminophenol (9, 9a), 2-(piperazin-1-yl)ethan-1-amine (10, 10a). Finally, the quinolinone residue was coupled to derivatives 2, 3, 4, 5, 6 and 7, as summarized in Supplementary Fig. S2, online to give the corresponding derivatives 2a-c, 3a-c, 4a-c, 5a-c, 6a-c and 7a-c. The reaction of pyrimidine 1a with IIc afforded compound 11 in which pyrimidine and dimethoxyquinolon-2-ylmethyloxy moieties are linked without any spacer between them.
Inhibitory effects on P-gp-mediated outward transport by the target compounds, cytotoxicity and SAR studies.
Flow cytometry findings showed that compounds 2, 2a-c, 3a-c, 4, 5, 8, 8a, 9 and 9a (Fig. 1), tested at 20 µM, were ineffective (p > 0.05) for increasing the intracellular accumulation of the fluorescent substrate Dox. However, compounds 3, 4a-c, 5a-c, 6, 6a-c, 7, 7a-c, 10, 10a and 11 (Fig. 1) at 20 µM significantly enhanced Dox retention with fluorescence intensity ratio (FIR) values ranging from 1.21 to 2.87, by efficiently blocking the efflux function of P-gp (Table 1). We therefore further evaluated these compounds at serial dilutions. As the results show in Table 1, compounds 7a, 10, 10a and 11 were able to induce a higher accumulation of Dox than the negative control, as from 1.25 µM (p < 0.05) followed by compounds 4c, 5b, 6c and 7c which showed minimum effective concentrations (MECs) of 0.62 µM (p < 0.05). Compounds 5c and 7b, were the most potent, as they still exhibited effectiveness at 0.31 µM (p < 0.05 and 0.001, respectively) (Table 1 and Fig. 2A and B). The efficacy of compounds 5c and 7b was similar to that observed with the classical P-gp inhibitor, verapamil, at all the concentrations assayed (p > 0.05), except at 0.31 µM (p < 0.05 and 0.01, respectively). The intracellular fluorescence-associated values in Lucena 1 cells treated with both compounds at 20 µM were similar to those observed in treated and untreated sensitive K562 cells (p > 0.05), which in turn showed no differences in their intracellular Dox fluorescence (p > 0.05) (Fig. 2C). These results suggested a complete reversal effect on P-gp-mediated MDR and a selective inhibition on P-gp function.
Table 1
Inhibitory effect of target compounds on P-gp transport activity in Lucena 1 and K562 leukemia cells
Compound
|
FIR Lucena 1
|
FIR K562
|
Concentration (µM)
|
Concentration (µM)
|
20
|
10
|
5
|
2.50
|
1.25
|
0.62
|
0.31
|
0.16
|
0.08
|
0.04
|
20
|
10
|
5
|
4a
|
1.21 ± 0.04***
|
1.03 ± 0.01
|
|
|
|
|
|
|
|
|
0.89 ± 0.03*
|
|
|
3
|
1.70 ± 0.22**
|
1.24 ± 0.02*
|
1.06 ± 0.05
|
|
|
|
|
|
|
|
0.98 ± 0.07
|
|
|
6a
|
1.40 ± 0.05***
|
1.30 ± 0.05*
|
1.15 ± 0.05*
|
1.06 ± 0.04
|
|
|
|
|
|
|
0.92 ± 0.04
|
|
|
7
|
2.23 ± 0.26***
|
1.42 ± 0.02*
|
1.19 ± 0.05*
|
1.07 ± 0.10
|
|
|
|
|
|
|
1.20 ± 0.06*
|
1.10 ± 0.08
|
|
4b
|
1.29 ± 0.08**
|
1.23 ± 0.08*
|
1.20 ± 0.06*
|
1.20 ± 0.06*
|
1.10 ± 0.13
|
|
|
|
|
|
0.90 ± 0.03**
|
|
|
5a
|
1.24 ± 0.07**
|
1.10 ± 0.02*
|
1.06 ± 0.01*
|
1.05 ± 0.01**
|
0.95 ± 0.05
|
|
|
|
|
|
0.86 ± 0.02*
|
|
|
6
|
1.65 ± 0.12***
|
1.38 ± 0.08*
|
1.18 ± 0.03*
|
1.18 ± 0.04*
|
1.09 ± 0.06
|
|
|
|
|
|
1.20 ± 0.02****
|
1.19 ± 0.02***
|
1.09 ± 0.04
|
6b
|
1.55 ± 0.07***
|
1.44 ± 0.13*
|
1.32 ± 0.09*
|
1.15 ± 0.05*
|
1.01 ± 0.03
|
|
|
|
|
|
0.94 ± 0.10
|
|
|
7a
|
1.33 ± 0.11*
|
1.52 ± 0.17**
|
1.38 ± 0.11*
|
1.28 ± 0.12*
|
1.10 ± 0.03*
|
0.95 ± 0.02
|
|
|
|
|
1.02 ± 0.07
|
|
|
10
|
1.72 ± 0.14**
|
1.30 ± 0.05*
|
1.41 ± 0.09*
|
1.26 ± 0.04**
|
1.11 ± 0.03*
|
1.06 ± 0.04
|
|
|
|
|
1.18 ± 0.03*
|
0.98 ± 0.06
|
|
10a
|
2.36 ± 0.25***
|
1.63 ± 0.17**
|
1.30 ± 0.08**
|
1.21 ± 0.02**
|
1.13 ± 0.03*
|
1.04 ± 0.03
|
|
|
|
|
1.26 ± 0.06**
|
1.13 ± 0.02**
|
0.98 ± 0.01
|
11
|
2.87 ± 0.55**
|
1.30 ± 0.07**
|
1.38 ± 0.07**
|
1.23 ± 0.04**
|
1.14 ± 0.02*
|
1.02 ± 0.02
|
|
|
|
|
1.44 ± 0.03*
|
1.01 ± 0.02
|
|
4c
|
1.39 ± 0.07**
|
1.28 ± 0.07*
|
1.32 ± 0.09*
|
1.31 ± 0.11*
|
1.24 ± 0.08*
|
1.11 ± 0.03*
|
1.03 ± 0.01
|
|
|
|
0.93 ± 0.02*
|
|
|
5b
|
1.36 ± 0.08***
|
1.25 ± 0.07*
|
1.31 ± 0.08**
|
1.29 ± 0.08*
|
1.21 ± 0.07*
|
1.16 ± 0.07*
|
1.07 ± 0.03
|
|
|
|
0.96 ± 0.02
|
|
|
6c
|
1.68 ± 0.12**
|
1.49 ± 0.20*
|
1.32 ± 0.09*
|
1.32 ± 0.08*
|
1.19 ± 0.07*
|
1.11 ± 0.03*
|
1.00 ± 0.01
|
|
|
|
0.79 ± 0.007**
|
|
|
7c
|
1.45 ± 0.10***
|
1.29 ± 0.11*
|
1.27 ± 0.04*
|
1.23 ± 0.02**
|
1.24 ± 0.05*
|
1.22 ± 0.07*
|
1.08 ± 0.05
|
|
|
|
1.04 ± 0.03
|
|
|
5c
|
1.42 ± 0.09**
|
1.24 ± 0.10*
|
1.23 ± 0.05*
|
1.26 ± 0.01**
|
1.22 ± 0.03*
|
1.29 ± 0.08*
|
1.12 ± 0.02*
|
0.99 ± 0.07
|
|
|
0.95 ± 0.02
|
|
|
7b
|
1.43 ± 0.10**
|
1.25 ± 0.04**
|
1.26 ± 0.04*
|
1.26 ± 0.04**
|
1.18 ± 0.03**
|
1.21 ± 0.01***
|
1.10 ± 0.01***
|
1.04 ± 0.02
|
|
|
0.88 ± 0.02*
|
|
|
Ver
|
1.55 ± 0.11****
|
1.38 ± 0.04****
|
1.34 ± 0.05***
|
1.31 ± 0.03***
|
1.29 ± 0.05***
|
1.22 ± 0.04**
|
1.23 ± 0.03***
|
1.14 ± 0.005***
|
1.11 ± 0.02*
|
1.09 ± 0.03
|
1.08 ± 0.05
|
|
|
Fluorescence intensity ratio (FIR) = mean fluorescence intensity (MFI) of Dox with compound/MFI of Dox alone. Ver: verapamil. Significant differences from the negative control were determined by using a paired one-tailed Student’s t test (****p < 0.0001, ***p < 0.001, ** p < 0.01, * p < 0.05).
|
With the exception of compounds 7c and 11 having IC50 values of 6.7 ± 0.20 and 3.8 ± 0.10 µM, respectively, against K562, all of the compounds showing modulatory activity were non-cytotoxic against the sensitive cells and the resistant counterpart as is evident from the IC50 values obtained, which were higher than 10 µM, the threshold established by the US National Cancer Institute for considering a compound as cytotoxic 3. The IC50 values for the non-toxic compounds were in all cases above 20 µM, except for compound 10a showing an IC50 value of 17.7 ± 0.15 µM against K562 and compounds 7, 7c, 10a and 11 with IC50 values of 17.9 ± 0.55, 10.2 ± 0.23, 17.1 ± 0.95 and 10.7 ± 0.15 µM, respectively against Lucena 1. Specifically, the most active compounds 5c and 7b were non-toxic, fulfilling the requisite of negligible cytotoxicity of the compound alone for developing effective P-gp modulators19,20.
Rhodamine 123 (Rho123) is a fluorescent probe efficiently effluxed by P-gp, which binds to the transporter at sites distinct from those of Dox5. Therefore, the ability of compounds 5c and 7b to enhance Rho123-associated intracellular fluorescence was further explored by flow cytometry. As shown in Fig. 3A and B, Lucena 1 cells treated with compounds 5c and 7b at 20 µM retained 2.72 ± 0.10 and 3.05 ± 0.16 -fold more Rho123 than the untreated cells (p < 0.01) with MECs of 0.62 µM (FIR = 1.36 ± 0.04 and 1.37 ± 0.06, respectively, p < 0.05). In comparison, both compounds increased the accumulation of Rho123 with efficiency (p > 0.05) similar to that of verapamil at 20 and 0.62 µM (FIR = 4.40 ± 0.82 and 1.22 ± 0.04). Meanwhile, no increase in Rho123 accumulation was observed in K562 (Fig. 3C), thus showing an effect ascribed only to inhibition of the P-gp extruding function.
It has been previously described that the number of hydrogen bond acceptor methoxy groups on terminal phenyl rings is favorable for P-gp inhibitory activity21–23. Reflecting this, compound 5c, bearing two OCH3 groups, displayed pronounced activity, followed by 5b with one methoxy and then compound 5a lacking this substituent (MECs = 0.31, 0.62 and 2.50 µM, respectively). The same trend was observed with compounds 7c, 7b and 7a (MECs = 0.62, 0.31 and 1.25 µM, respectively) (Table 1). It is important to highlight that the instability of the dimethoxy compound 7c may have slightly masked its anti-P-gp effect, therefore showing a higher minimum effective concentration (MEC) value than that expected in comparison to compound 7b, feathered with only one OCH3. Increasing the number of methoxy substituents also resulted in improved activity in the chlorinated compounds 4c and 4b compared to compound 4a (MECs = 0.62, 2.5 and 20 µM, respectively) (Table 1). The same was observed with the chloride compounds 6c and 6b with respect to compound 6a (MECs = 0.62, 2.5 and 5 µM, respectively). The presence of the p-chlorophenyl group was detrimental to the inhibitory effect of compounds 4a-b (MECs = 20 and 2.5 µM, respectively) and 6a-b (MECs = 5 and 2.5 µM, respectively) in comparison to their respective analogues 5a-b and 7a-b which instead bear a naphtha-2-yl group. Interestingly, the presence of two OCH3 groups counteracts the adverse effect exerted by the chlorobenzene substituent, as was observed when compounds 4b and 6b (MECs = 2.5 µM) were compared with their respective analogues 4c and 6c, showing MEC values of 0.62 µM, the latter with similar activity to that of the closely related compounds 5c and 7c, respectively (Table 1). It has been previously reported that compounds featuring chlorine at ortho-, meta- and para-positions, also bearing isoquinolines with two methoxy groups, exhibited similar P-gp modulatory activity to that observed with verapamil11. The results obtained clearly indicated that the methoxy substituents significantly influenced P-gp inhibition. The replacement of the p-phenylenediamine in compounds 4a-c and 5a-c for a carbonyl group as in the respective compounds 2a-c and 3a-c prevented the anti-P-gp effect, regardless of the presence of OCH3 or chloride substituents (Table 1). The clear loss of activity of these compounds was further revealed by docking and molecular dynamics (MD) simulations.
Reversal activity on doxorubicin toxicity of the most promising compounds 5c and 7b.
To validate the assumption that increased accumulation of Dox is associated with a concomitant enhancement in its cytotoxicity, compounds 5c and 7b were co-administered with this chemotherapeutic drug and the effect on cell viability was determined. Consistent with the accumulation of Dox, compounds 5c and 7b at 1.25 µM substantially decreased the half-maximal inhibitory concentration (IC50) values of Dox (Fig. 4), circumventing Lucena 1 resistance with fold reversal (FR) values of 5.16 ± 0.60 and 6.90 ± 2.84, respectively, and showing a similar potency to that of verapamil (p > 0.05) with a FR value of 6.21 ± 1.28. Compound 5c was still able to reverse Dox resistance when the concentration decreased to 40 nM (FR = 1.30 ± 0.05), while compound 7b still chemosensitized Lucena 1 at 80 nM (FR = 1.51 ± 0.23) (Fig. 4). The activity observed with 5c and 7b was not significantly different (p > 0.05) than that of verapamil (FR = 1.55 ± 0.17 and 1.40 ± 0.11 at 40 and 80 nM, respectively).
Molecular modelling.
With the aim of gaining insight into the way that ligands interact with P-gp, molecular modeling was performed. The protocol for this analysis was divided into three interrelated parts with increasing levels of detail. First, in the docking protocol, the interior of the whole transmembrane region was scanned, making no assumptions about the localization of a particular binding site. The main binding sites were determined for the experimental reference chemotherapeutic drug, Dox, the known reference inhibitors tariquidar and verapamil and the subject compounds shown in Fig. 1. Next, detailed analyses were performed of the binding dynamics of the selected species with poor, mild or high activity, and an estimate of each free energy of binding was calculated by means of classical MD simulations. The trajectories obtained were also analyzed to identify the main contributions to these by each residue. Finally, each interaction was discussed in more detail in the light of the QTAIM analyses of hybrid QM/MM calculations.
The primary binding region for the experimental reference chemotherapeutic drug, Dox, was found to be overlapped with the region of the bulkier cytotoxic agent paclitaxel (Taxol), which was co-crystallized in the novel P-gp structure (Fig. 5)24. The docking results started to shed light on the differences in the activities of the panel of assayed compounds, even for those having similar structures but sharp differences in Dox accumulation profiles. Most of the mild and all the powerful inhibitors fell inside the site that was also shared by Dox and Taxol (Fig. 5), involving strong interactions with the aromatic residues, mainly from TMH 4, 5 and 6 from one homologous half and with those from TMH 7 and 12 from the other half of the transporter. Figure 6A shows a superimposition of the most stable docked poses of active compounds 4b, 5b-c, 6a-b, 7a-c, 10 and 11 along with the top reference inhibitor, tariquidar and Dox. A gross common feature for all these poses in this binding pocket is that they all fold into a distorted “U”-shape. The aromatic edges of each compound accommodated as the branches of the “U” with an aliphatic/conjugated flexible bridge as its camber.
This flexible intermediate portion, which also itself interacts with TMH 12, allowed for the turn or camber. In contrast, the compounds depicted in Fig. 6B are those that were too short or too long and not flexible enough to accommodate as the more powerful compounds did at the site of Fig. 6A. None of them were active. These molecules can either fit into just one of the branches of the “U”, as observed with compounds 5, 8a or 9a, or fall into a different pocket where there is no interaction with residues from TMH 6 and 12, as observed with compounds 2, 2b, 2c, 3a-c, 8 or 9. The relevance of such structural determinants may be clearly illustrated by comparing the inactive compound 3c with one of the strongest inhibitors, compound 5c, both with very similar chemical structures, including the ortho-dimethoxy substitution in the quinolinone ring. The only difference between both entities is that the flexible linker to the quinolinone, the group —NHCH2—, is replaced by a longer and less flexible carbonyl-vinyl (—C(O)—CH═CH—) (Fig. 1). In addition to the two main groups of compounds shown in Fig. 6A and B, compound 3 with moderate activity (MEC = 10 µM, Table 1) was found to fit partially into the pattern of Fig. 6A, also touching TMH 11, a contact that is not shared by any of the other ligands (Supplementary Fig. S3, online).
While the docking did reveal some clear trends in the type of interaction and sites of relevance, MD simulations provided a more detailed understanding of the nature, strength, and persistence of interactions with the key residues. The inactive compounds 5 and 8, with the patterns shown in Fig. 6B, were selected for the studies, as well as the active compounds 3, 5a, 5c and 7b, the respective activity of which increased from low to mild to the highest (Table 1). The free energies of binding (ΔG0b) of these representative compounds, estimated by MD, were compared to those of the substrates and of the two reference inhibitors, verapamil, used as a positive control, and tariquidar, one of the most potent in vitro inhibitors known2, used as a positive control for the in silico studies. Although one substrate and tariquidar and verapamil were previously simulated8,25, for comparison purposes, these simulations were repeated from scratch with the novel PDB structure and using exactly the same simulation conditions as for the subject ligands. As shown in Table 2, compounds 5 and 8 were found to be unable to compete with either Rho123 or Dox, added to the fact that compound 8 went to another site of lower affinity. The ΔG0b values of compounds 3, 5a, 5c and 7b (Table 2) reproduced well their inhibitory properties (Table 1). The most promising structures, 5c and 7b, showed similar energies to that of verapamil although not as favorable as tariquidar (Table 2). The latter comparison was not surprising, since tariquidar is known to have a low nanomolar activity26, despite that its high toxicity27, in contrast to our subject compounds, and other issues28 prevent it being clinically suitable.
Table 2
Free energies of binding from the MMPBSA analyses of the MD simulations.
Compounds
|
ΔG0b (kcal/mol)
|
Substrates
|
|
rhodamine 123
|
-23.2
|
doxorubicin
|
-24.2
|
Subject compounds
|
|
5
|
-16.7
|
8
|
-22.8
|
3
|
-27.1
|
5a
|
-29.4
|
5c
|
-33.1
|
7b
|
-35.8
|
Reference inhibitors
|
|
verapamil
|
-35.7
|
tariquidar
|
-38.3
|
An analysis of the dynamic interaction of the potent compound 7b throughout the simulation revealed that it persistently shared the region and the contacts of the known therapeutic P-gp substrates, Dox and Taxol. In contrast, the inactive compound 8, besides its reduced affinity in terms of ΔG0b, stayed in a different region and had greater mobility as shown in Fig. 7, where four representative snapshots of each MD trajectory (i.e. the conformations most visited at the simulation temperature) are superimposed for Dox, Taxol and compounds 7b and 8. A similar behavior was observed for compounds 5a and 5c. Figure 8A-C shows the ΔG0b components decomposition in terms of per residue energy for compound 5c, compared to the substrate Dox and tariquidar. Most of the contacts of 5c were shared with the top reference inhibitor. More important is the fact that most of these contacts, such as
Ala229 and Trp232 from TMH 4, Phe303 and Ile306 from TMH 5, Ile340, Phe343 and Gln347 from TMH 6, and Phe983 and Met986 from TMH 12, among others, were proposed to play a key role in the binding to P-gp on both computational and experimental bases5,8,25,29. Compound 5c (Fig. 8C), as well as compound 7b (Supplementary Fig. S4, online) showed important peaks (i.e. persistent contacts) at residues from both homologous halves (TMHs 1–6 and 7–12), in particular from TMH 6 and 12, both connecting the transmembrane domain (TMD) to the nucleotide binding domains. As depicted in Fig. 8D, and as previously observed in the docking poses, the inactive compound 5 shared most of the contacts present in TMH 5 and 6 with compounds 5c (Fig. 8C) and 7b (Supplementary Fig. S4, online) but did not interact with those from TMH 12 or with any other from the second homologous half. This energy analysis also revealed a certain importance of contact with TMH 7 (mainly with Gln725), which is observed with compounds 5a, 5c and 7b as well as with tariquidar, but not with Dox or the less active compound 3 or the inactive compound 5 (Figs. 8 and Supplementary S2, online). Indeed, compounds 3 and 5 contact with neither TMH 7 nor TMH 12; the latter only contacted with the second half with a mild interaction with Gln946 from TMH 11 (the decomposition profile for compounds 3, 5a, 8 and verapamil are shown in Supplementary Fig. S4, online).
The MD simulations also revealed that the number of hydrogen bond acceptors in the molecule would not be as important as the other features discussed so far. Indeed, even though tariquidar is the species with the most H-bond acceptors, it just had a total average of about one H-bond during the simulation time; the same was observed for compounds 5c and 7b. On the other hand, the compound which kept the highest number of persistent H-bonds during the simulation was 3, one of the poorest of the set (see Supplementary Fig. S5, online for a comparison of the H-bond analyses over the last 10 ns of simulations for 3, 5c, 7b and tariquidar). The main interactions with the residues bearing the major peaks in Fig. 8 are hydrophobic in nature and are analyzed in more detail with QM/MM calculations.
Charge density analysis of complexes.
QTAIM calculations are very useful tools for evaluating accurately and in detail the molecular interactions which stabilize ligand-receptor complexes30 and can be used successfully in different biological systems31–35. In order to analyze the molecular interactions stabilizing the complexes of the target molecules and P-gp more quantitatively and with more detail, QM/MM calculations were performed, choosing the most representative complexes of the series. Therefore, the complexes of Dox, tariquidar and compounds 3, 5a, 5c, 7b and 8 with P-gp were further studied by Charge Density Analysis in the context of the quantum theory of atoms in molecules (QTAIM) framework. Reduced models of complexes were constructed, containing receptor residues within the range of noncovalent interactions from ligand atoms, as reported in the experimental section. The QTAIM topological analysis was performed by mapping the gradient vector field onto the pre-computed charge density (i.e., Δ⍴(r)) of reduced models, thus giving rise to the topological elements of the charge density, i.e., the bond critical points (BCPs) and the bond paths (BPs) that connect the interacting atoms.
Figure 9 shows the charge density values obtained for the complexes in function of the different TMH domains of P-gp, and the numerical data is shown in Supplementary Table S1, online. It is interesting to note that the complexes with Dox and the inhibitors tariquidar, 5a, 5c and 7b presented a very similar binding pattern, with all the significant interactions located in residues from TMH 4–6 and 12, the most important of which were with TMH 6 and 12. It is important to highlight that tariquidar as well as compounds 5c and 7b, and to a lesser extent compound 5a, showed significant interactions with TMH 7. Weaker interactions were also seen with residues located at TMH 1 and TMH 10, while no interactions with TMH 2 were observed in any complex. One exception to this motif was the case of compound 5c, which showed somewhat significant interactions with amino acids from TMH 10. Regarding the mildly effective compound 3, the pattern of interactions was different from that observed for the active compounds since it showed the strongest interactions with TMH 6 but lacked interactions with TMH 12. In addition, it was the only compound displaying interactions with TMH 11. The inactive compound 8 did not establish interactions with the residues of either TMH 6 or TMH 7, although it interacts with TMH 12, but with different residues to those which compounds 5c and 7b contacted. Likewise, 3 was the only compound that established interactions with TMH 8 and TMH 9. Therefore, the low or null activity of compounds 3 and 8 may be explained by their binding in widely different sites.
Besides the benefits of QTAIM as a tool for describing molecular interactions through visual inspection of the charge density in molecular graphs, it has been shown that there is also a direct relationship between the charge density value in the BCP interaction and the complex interaction energy36. Therefore, one might expect this relationship to still hold for biomolecular complexes, i.e., the sum of charge density values from all the intermolecular BCPs should be related to the complex stability. Such a relationship concerning a particular system is important since it enables the contributions of individual functional groups to the overall anchoring strength of the ligand within the binding pocket to be measured. In order to clarify the interactions from the point of view of the target ligands, the molecules were arbitrarily divided into three portions (moiety 1, linker and moiety 2), as shown in Fig. 10. Figure 11 shows the total of interactions established according to the three portions of each molecule. Compounds 3 and 8 were not included because these interacted in a different site and, therefore, comparing their interactions with active compounds would be futile. All the complexes showed a similar pattern of interactions and indicated that the three portions of the ligands establish significant interactions, stabilizing the complexes. In other words, it appears that the presence of the three portions of the ligands is necessary for the activity. As expected, the interactions observed for the complex with tariquidar were the strongest while interactions obtained for the complex of 5a were the weakest, while the interactions obtained for Dox, 5c and 7b complexes gave intermediate values. These results fully match both the experimental data and the classical MD estimations.
Figure 12 shows the molecular interactions between Dox and P-gp. As can be seen, Dox mainly interacts with residues from TMH 6 (Phe343, Ser344 and Gln347) and TMH 12 (Met986 and Gln990). Atoms belonging to the naphthoquinone ring (moiety 1) are stacked between the aromatic residues Trp232, Phe303 and Phe343 (TMH 4, TMH 5 and TMH 6 respectively), establishing several π-stacking type interactions. These interactions certainly contributed to the overall anchoring of this ligand in the active site as it favorably oriented the rest of the molecule to interact with TMH 6 and TMH 12. Particularly, OH groups and the hydroxyacetyl substituent from the linker were hydrogen-bonded to neighboring polar residues such as Ser196 (TMH 3), Ser344, Gln347 (TMH 6) and Gln990 (TMH 12). Moreover, moiety 2 significantly interacted with Met986 and Gln990 and also established hydrophobic interactions with Leu65 (TMH 1) and Ile340 (TMH 6). On the other hand, the larger size and greater flexibility of tariquidar compared to Dox prevented the latter from establishing the same interactions. In the case of tariquidar, interactions with TMH 1, TMH 5 and TMH 7 were significantly increased while those with TMH 3, TMH 10 and TMH 12 were reduced or even not present at all (Fig. 9).
Although the quinoline ring was still anchored over the aromatic residue Trp232, unlike the moiety 1 of Dox for which the main stabilizing interactions were with TMH 4, TMH 5 and TMH 6, the modeling of tariquidar suggested that this portion interacted especially with TMH 5 (Fig. 13). As observed in this Figure, the quinoline ring formed several hydrophobic interactions with Leu236 from TMH 4, and with Ile299 Phe303 and Ile306 from TMH 5. In addition, several interactions of the linker were formed with hydrophobic and aromatic residues from various TMH. The presence of two aromatic rings and two methoxy substituents in the linker also increased the possibility of hydrophobic interactions. The strongest interactions of this moiety were with Leu65 (TMH 1), Tyr307 (TMH 5), Ile340 and Phe343 (TMH 6), Phe728 (TMH 7) and Phe983 (TMH 12), while a single H-bond was also observed between Gln347 (TMH 6) and the amide carbonyl oxygen bound to moiety 1.
This result supports the idea that the biological effects of the compounds studied here may not be mainly related to the possibility of forming H-bonds but depend on other structural features. Regarding moiety 2, while Dox was almost completely bounded through strong interactions to residues from TMH 12, tariquidar displayed a different pattern. This formed several interactions with TMH 5 (Phe303 and Tyr307), TMH 7 (Leu724) as well as with TMH 12 (Ala987, Gln990 and Val991). Thus, charge density value was significantly higher in the tariquidar complex. In addition, although interactions with TMH 12 were weaker compared to Dox, the charge density regarding TMH 12 remained high as tariquidar establishes a greater number of interactions. Active compound 5c showed some similarities with both Dox and tariquidar, as well as some differences. It must be pointed out that moiety 1 is not located in the same region discussed for the other compounds. In fact, moiety 1 of compound 5c did not interact with Trp232, which could explain in part the low ρ(r) value observed for the naphthyl ring (see blue zones of Fig. 10). Nevertheless, strong interactions were observed between moiety 1 and TMH 5 and TMH 6, and in particular with Phe303, which resemble those observed for Dox. In addition, even though the conformation adopted by 5c prevented it from effectively interacting with Trp232, strong π-stacking type interactions with Phe343 (TMH 6) were formed. The anchoring loss from moiety 1 is compensated by the significant interactions established by the linker. As can be seen in Fig. 14, residues Gln347 (TMH 6) and Glu875 (TMH 10) were H-bonded to both NH groups of the compound 5c linker moiety. Also, hydrophobic interactions with Ala229 (TMH 4) and C-H···π interactions between the pyrimidine ring, located at the linker zone, and the aromatic Trp232 were formed. In addition, the linker established CH2···SCH3 and Ar-H···NH2 interactions with Met986 and Gln990, respectively, both from TMH 12. Finally, moiety 2 interacted with TMH 12 (i.e., Ala987 and Gln990) in a similar way to that discussed for tariquidar. However, Fig. 14 also shows that a significant interaction occurred with TMH 7, especially with Gln725, which is H-bonded to methoxy groups from compound 5c.
Toxicity assessment of most active compounds. As the most potent molecules, the cytotoxicity was determined of compounds 5c and 7b against normal peripheral blood mononuclear cells (PBMC), in an effort to evaluate their safety profile. The results showed that compounds 5c and 7b were devoid of cytotoxicity, with 21% and 12% of inhibition on proliferation at 20 µM, respectively. In addition, compounds 5c and 7b did not alter the erythrocyte membrane at concentrations lower than 10 and 20 µM, respectively. The results show that these compounds may be proposed as safe candidates for developing novel agents to circumvent MDR by P-gp inhibition.