3.1. CoMFA and CoMSIA Models
The statistical parameters for the CoMFA and CoMSIA models are given in
Table 3. For the CoMFA model, partial least squares (PLS) regression produced a excellent cross-validated correlation coefficient (
r2cv) of 0.726 (>0.5) with an optimized component of 6, which suggesting that the model is reliable and it should be a useful tool for predicting the IC
50 values. The non cross-validated PLS analysis gave a high correlation coefficient (
r2) of 0.972,
F value of 105.300 and a low standard error estimate (SEE) of 0.144. The contributions of steric and electrostatic fields to this model were 0.457 and 0.543, respectively. The predictive correlation coefficient (
r2pred) value based on molecules of the test set was 0.937 for the CoMFA model. The actual and predicted pIC
50 values of the training set and test set by the model are given in
Table 2. The relationship between actual and predicted pIC
50 of the training set and test set compounds of the CoMFA model is illustrated in
Figure 3a, where almost all points are located on the diagonal line.
For the CoMSIA model, the statistical parameters revealed that steric, electrostatic, hydrophobic, hydrogen bond donor and acceptor features significantly influence the activity of the inhibitors. The CoMSIA model gave a cross-validated correlation coefficient (
r2cv) of 0.566 (>0.5) with an optimized component of 6, which suggested that the model is reliable and should be a useful tool for predicting the IC
50 values. The non cross-validated PLS analysis gave a high correlation coefficient (
r2) of 0.984, F value of 181.398 and a low error estimate (SEE) of 0.110. The contributions of steric, electrostatic, hydrophobic, hydrogen bond donor and acceptor fields were 0.152, 0.169, 0.243, 0.183 and 0.252, respectively. The predictive correlation coefficient (
r2pred) value based on molecules of the test set was 0.948 for the CoMSIA model. The actual and predicted pIC
50 values and residual values for training set and test set compounds are given in
Table 2. The graph of actual activity
versus predicted pIC
50 of the training set and test set is illustrated in
Figure 3b, where almost all points are located on the diagonal line.
3.2. CoMFA and CoMSIA Contour Maps
The results of the CoMFA and CoMSIA models were visualized through contour maps. These maps showed regions in 3D space where variation in specific molecular properties increased or decreased the activity. The molecular fields around the most active compound
20 are displayed in
Figures 4–
6, accordingly. These contour maps are significant for drug design, as they showed regions in 3D space where modifications of the molecular fields strongly correlated with concomitant changes in biological activity.
The steric contour map of CoMFA is shown in
Figure 4a, which was almost the same as the corresponding CoMSIA steric contour map (
Figure 4b). Compound
20 was selected as a reference molecule. The steric field was represented by green and yellow contours, in which green contours indicate regions where presence of bulky steric groups was favored and should enhance inhibitory activity of molecules, while the yellow contours represent regions where occupancy of steric groups was unfavorable. As shown in
Figure 4, the presence of the green contour around the R
1 position suggested that a bulky group at this region would be favorable. By checking up all the R
1 modified compounds, it was found that derivatives
07–
08 have the activity order of
07 (R
1 = Br) >
08 (R
1 = NO
2); compounds
13,
14,
17 have the activity order of
14 (R
1 = −SO
2CH
2CHCH
2) >
13 (R
1 = −SO
2C
2H
5) >
17 (R
1 = −SO
2NH
2); compounds
17–
19 have the activity order of
20 (R
1 = sulfo-pyrrolidine) >
19 (R
1 = −SO
2N(CH
3)
2) >
18 (R
1 = −SO
2NHCH
3) >
17 (R
1 = −SO
2NH
2); compounds
23–
26 have the activity order of
23 (R
1 = −NHSO
2C
2H
5) <
24 (R
1 = −NHSO
2-benzene),
25 (R
1 = −NHSO
2-CH
2-benzene) <
26 (R
1 = −NHSO
2-benzene). These were satisfactory according to the steric contour map. The R
2 was surrounded by three yellow contours, which suggested a bulky group at this region would decrease the inhibitory activity. This may explain why compounds
1–
2,
5, which possessed a relative bulky group (e.g., −COOEt) at R
1, showed significantly decreased activities than other compounds with a relatively minor substituent at R
2. For instance, derivative
24 bearing a carboxy group at R
2 exhibited improved potency than compound
26 with an ethoxycarbonyl at this position. Furthermore, compound
20 with carboxyl group at the R
2 position was the most inactive compound.
The electrostatic field contour maps of CoMFA and CoMSIA are shown in
Figure 5a and b, respectively. Compound
20 was selected as a reference molecule again. The electrostatic field is indicated by blue and red contours, which demonstrate the regions where electron-donating group and electron-withdrawing group would be favorable, respectively. In the electrostatic field, two blue contours around the terminal of R
1 and two red contours at the middle of the R
1 revealed that the electron-donating substituents at the terminal of the R
1 and the electron-withdrawing groups at the middle of the R
1 were essential for the inhibitory activity. Take the compounds
13–
22 and
24–
26 (R
1 = 4-CF
3-benzyl) for an example, the strong electron-withdrawing sulfo, and sulfanilamido group at the middle of R
1 and electron-donating Et, −NH
2, −NHCH
3, −N(CH
3)
2, 1-pyrrolidine, 1-piperidine, and 4-morpholine groups at the terminal of R
1 in these compounds resulted in significantly increased activity. The blue contour near the chain between 6,7-dihydro-1
H-inden-4(5
H)-one and R
2 suggested the electron-donating group (-CH
2CH
2-) at this position may be essential for potency. All of the derivatives involved in this study possessed a -CH
2CH
2- group at this site, which revealed the extreme importance of the electron-donating substituent.
In hydrophobic fields, yellow and white contours highlighted areas where hydrophobic and hydrophilic properties were favored. In
Figure 6a, the yellow contour around the chain of R
1 position indicated that a hydrophobic substituent would benefit the potency. Most of the derivatives involved in this study possessed a hydrophobic group at this site, which revealed the extreme importance of the hydrophobic substituent. Furthermore, the actual IC
50 of these compounds was basically 10-fold than those without a hydrophobic group. A huge white contour near the terminal of R
1 site suggested that a hydrophilic group may be favored. This may explain why derivative
20 with a relatively more hydrophilic N atom at this position exhibited better potencies than compounds
25–
33. Another white contour around R
2 position demonstrated that a hydrophilic substituent carboxyl would be favorable. Most of the compounds possessed a hydrophilic substituent at this site, except compounds
1–
2 with a hydrophobic −OOC
2H
5 at R
2, which displayed lower activity than compounds
3–
25.
In hydrogen bond donor field, the cyan and purple contours indicated favorable and unfavorable hydrogen bond donor groups. In
Figure 6b, the two bulk purple contours near the R
1 position revealed that hydrogen bond acceptor groups may benefit the potency. In fact the sulfo group and quaternary amine atom at this position acted as hydrogen bond acceptor by forming H-bonds with residues of the ATP binding site of Aurora A. This may explain why compounds
19–
22 displayed relatively better activities. Likewise, a bulk purple contour near R
2 revealed that they acted as hydrogen bond acceptor by forming H-bonds with residues of the ATP binding site of Aurora A. Most of the derivatives involved in this study possessed a hydrogen bond acceptor group (carboxyl) at this site, which indicated the extreme importance of the hydrogen bond acceptor group substituent.
In hydrogen bond acceptor field, the magenta and red contours identified favorable and unfavorable positions for hydrogen bond acceptors. In
Figure 6c, two bulk red contours around the R
1 and R
2 indicated that a hydrogen bond acceptor substituent at these sites would increase the activity. The inference obtained by
Figure 6c satisfactorily matched the hydrogen bond donor contour map.
3.3. Docking Analysis
Surflex-Dock was applied to investigate the binding mode between these pyrrole-indoline-2-ones and Aurora A. In this paper, Surflex-Dock could also serve to inspect the stability of 3D-QSAR models previous established. To visualize secondary structure elements, the MOLCAD Robbin surfaces program was applied. Furthermore, the MOLCAD surface of ATP site was also developed and displayed with cavity depth (CD), electrostatic potential (EP) to further explore the interaction between these inhibitors and the receptor. The most potent inhibitor 20 was selected for more detailed research.
In
Figure 7a, the hydrogen bonding (dashed lines) interactions between the reference compound
20 with highest inhibitory activity is shown and the key residues (Lys162, Asp274, Glu211, and Arg220) of the ATP site of Aurora A (PDB code 2X6E) are labeled. A total of five hydrogen bonds were formed: the sulfo at R
1 position acted as the hydrogen bond acceptor and formed two H-bonds with the secondary amino group of the Tys162 residue, and a H-bond with the secondary amino group of Asp274; the carbonyl substituent on the 6,7-dihydro-1
H-inden-4(5
H)-one scaffold in compound
20 also acted as the hydrogen bond acceptor and formed H-bond with primary amino group of Arg220 residue; the secondary amino on the 6,7-dihydro-1
H-inden-4(5
H)-one in compound
20 acted as H-bond donor and formed H-bond with carbonyl group of Glu211. These results observed by
Figure 7a satisfactorily matched the observation taken from the CoMSIA hydrogen bond donor contour map.
In
Figure 7b, the secondary structure of the ATP pocket within compound
20 is depicted: alpha helices are displayed as helices or cylinders, while beta sheets are shown as arrows and the loop regions as tubes. The key residues and hydrogen bonds (dashed lines) are labeled.
In
Figure 7c, the MOLCAD Multi-Channel cavity depth potential surfaces structure of the binding site within the compound
20 is displayed and the cavity depth color ramp ranged from blue (low depth values = outside of the pocket) to light red (high depth values = cavities deep inside the pocket). In
Figure 7c, the R
1 position of compound 20 is observed in a blue area, revealing that this position was embedded deep inside the ATP pocket. It can be simply inferred that a bulky group at R
1 position may be favorable. Since the R
2 site was oriented to a light red area, which illustrated a minor group was anchored into a favorable region, this suggests that minor groups may benefit the potency. The observation obtained by
Figure 7c satisfactorily matched the corresponding CoMFA and CoMSIA steric contour maps.
In
Figure 7d, the MOLCAD electrostatic potential surface of the binding region was demonstrated with the color ramp for EP ranging from red (most positive) to purple (most negative). The 1-pyrrolidinyl group at the terminal of R
1 was found in a blue area, which indicated that electron-donating properties at this site were essential for the potency; the sulfo group was in a yellow area, which suggested that electron-withdrawing properties would be favored; the -CH
2CH
2- chain between 6,7-dihydro-1
H-inden-4(5
H)-one and carboxyl was anchored in a blue area which suggested that an electron-donating substituent at this position would be essential for the potency. These results were well compared with the corresponding CoMFA and CoMSIA electrostatic contour maps.
3.4. Design for New Molecules
The detailed contour map analysis of both COMFA and CoMSIA models and the docking analysis empowered us to identify structural requirements for the observed inhibitory activity (
Figure 8). In detail, bulky, electron-donating, hydrophobic, and hydrogen bond acceptor substituents at the terminal of R
1 (e.g., pyrrolidine) would increase activity; bulky, electron-withdrawing, hydrophilic, and hydrogen bond acceptor substituents at the chain of the R
1 (e.g., sulfo group) are favored for inhibitory activity; minor, hydrophilic, hydrogen bond donor groups at the R
2 (e.g., carboxyl) may benefit potency. Moreover, the electron-donating chain (-CH
2CH
2-) between pyrrole and R
2 may be essential for the activity of the inhibitors. The chain of the R
1, indolin-2-one, and 6,7-dihydro-1H-indol-4(5
H)-one groups were crucial for binding to ATP pocket of Aurora A.
Based on QSAR and docking results, inhibitor
20, with the highest activity, was taken as a template to design new compounds. A set of nine new compounds with high predicted activity were designed and assessed (
Table 4), and the graphs of their predicted pIC
50 values
versus the most active compound
20 are shown in
Figure 9. After energy minimization, the nine new compounds were docked into the ATP binding site of Aurora A. The total scores of these compounds were higher than that of the template molecule (
Table 4). The designed molecule
D8 was selected for more detailed investigation as one more H-bond from carbonyl on indolin-2-one group with Ala213 can be observed in
Figure 10, where the compound
D8 with the highest surflex-dock total score were docked into the ATP pocket of Aurora A.