3.1 Validation of molecular modelling protocols
Nineteen structures (see Table 1) of coordinates for hMcl-1 co-crystallized with ligands from the protein data bank (www.rcsb.org) were retrieved. Although these crystal structures belong to the same species (Homo sapiens, h), their quality varied. Validation metrics are shown in Table 1, which are independent of the ligand. There were hMcl-1 crystal structures that contained peptide ligands (Table 1, entries 10–19), and these showed typical interactions with amino acids of the cavity, including those in the pockets: P1, P2, P3, and P4 (Fig. 1). We did not proceed further study with those that had peptide ligands, which interacted with P1-P4. Small molecule inhibitors (SMIs) co-crystallized (Table 1, entries 1–9) with hMcl-1 have a wider diversity of scaffolds compared to peptides, with a greater variety of binding modes. It was decided that these nine SMI structures would be used to generate binding modes for the natural products.
Table 1
Parameters of co-crystal structures from the protein databank where hMcl-1 binds to a ligand [30, 31].*
Entry
|
PDB code
|
Res. (Å)
|
Co-crystallized ligand type
|
R-value
|
R-free
|
Clash score
|
Ramachandran outliers (%)
|
Sidechain outliers (%)
|
RSRZ outliers
(%)
|
Reference
|
1
|
4WMR
|
1.70
|
SMI*
|
0.171
|
0.206
|
1
|
0
|
0
|
4.0
|
[32]
|
2
|
4ZBF
|
2.20
|
SMI
|
0.184
|
0.233
|
6
|
0.2
|
4.5
|
2.0
|
[33]
|
3
|
4ZBI
|
2.50
|
SMI
|
0.183
|
0.242
|
7
|
0.6
|
8
|
1.7
|
[33]
|
4
|
4OQ5
|
2.86
|
SMI
|
0.200
|
0.235
|
9
|
2.1
|
11.3
|
8.1
|
[34]
|
5
|
4OQ6
|
1.81
|
SMI
|
0.203
|
0.205
|
11
|
0
|
7.3
|
1.4
|
[34]
|
6
|
3WIX
|
1.90
|
SMI
|
0.246
|
0.291
|
4
|
0
|
3.3
|
3.2
|
[35]
|
7
|
3WIY
|
2.15
|
SMI
|
0.214
|
0.283
|
3
|
0.7
|
4.0
|
2.5
|
[35]
|
8
|
4HW2
|
2.80
|
SMI
|
0.217
|
0.251
|
32
|
1.7
|
17.6
|
1.8
|
[36]
|
9
|
4HW3
|
2.40
|
SMI
|
0.216
|
0.269
|
13
|
1
|
11.4
|
3.2
|
[36]
|
10
|
4HW4
|
1.53
|
Peptide
|
0.140
|
0.182
|
2
|
0
|
0
|
2.1
|
[36]
|
11
|
3TWU
|
1.80
|
Peptide
|
0.184
|
0.223
|
2
|
0
|
0
|
2.3
|
[37]
|
12
|
3PK1
|
2.49
|
Peptide
|
0.213
|
0.245
|
3
|
0.9
|
8.1
|
0.9
|
[38]
|
13
|
3MK8
|
2.32
|
Peptide
|
0.233
|
0.275
|
9
|
0.7
|
0
|
1.9
|
[39]
|
14
|
3KZ0
|
2.35
|
Peptide
|
0.224
|
0.270
|
10
|
0
|
0
|
9.9
|
[40]
|
15
|
3KJ0
|
1.70
|
Peptide
|
0.187
|
0.223
|
8
|
0
|
0.6
|
4.4
|
[40]
|
16
|
3KJ1
|
1.95
|
Peptide
|
0.188
|
0.213
|
3
|
1.2
|
0.7
|
9.4
|
[41]
|
17
|
3KJ2
|
2.35
|
Peptide
|
0.210
|
0.233
|
2
|
0
|
1.3
|
6.7
|
[41]
|
18
|
3IO9
|
2.40
|
Peptide
|
0.211
|
0.269
|
7
|
0.6
|
4.9
|
4.1
|
[42]
|
19
|
2PQK
|
2.00
|
Peptide
|
0.196
|
0.234
|
6
|
0
|
1.4
|
7
|
[42]
|
*SMI = small molecule inhibitor. PDB = ID code from the protein data bank (www.rcsb.org); Res: Resolution; R-Value: a measure of how a simulated diffraction pattern matches the experimentally-observed diffraction pattern, a perfect fit would have a value of 0 and, typical values are about 0.20; R-free: 10% experimental observations were removed from the data set and then refinement is performed using the remaining 90%, the R-free value is calculated from how well the model predicts the 10% of observations that were not used in refinement, R-free is typically found to be slightly higher than R-value and with a value of close to 0.26; Clash Score: the number of pair of atoms clashing sterically per 1000 atoms; Ramachandran outliers: fraction of amino acids with non-favorable dihedral angles. Sidechain outliers: off-rotamer positions or less stable conformations of amino acid side chains compared to the on-rotamer position or statistically more favored conformation. The score is calculated as the percentage of residues with unusual sidechain conformation to the total number of amino acid residues; RSRZ outliers: an amino acid residue is said to be an RSRZ outlier if its RSRZ value is more than two. The RSRZ outlier score depends on the fraction of polypeptide residues that do not fit electron density well when compared with other instances of the same residues in structures at similar resolution. [43]; RMSD: root mean square deviation. Here this is a measure of the average distance between the atoms (usually the backbone atoms) of docked conformation of SMI to the conformation in the crystal structure. |
Having selected nine co-crystal structures of hMcl-1 with SMIs, the multiple receptor conformations-based approach (MRC) [44] was implemented, using the ensemble of conformations from the crystal structures (Fig. 3a). Thus, the backbone atoms of these conformers were superimposed (entries 1–9, Table 1, excluding the small molecules), with subsequent calculation of the various RMSD values (Table 2). The hMcl-1 backbone conformation was found to be conserved based on RMSD values between 0.52 to 1.13 Å; these values were considered acceptable given the total number of amino acids (157) in the protein backbones (Table 2). The average RMSD values are also reported.
Next, the multiple ligand conformations-based approach (MLC) was applied, which involved docking all SMIs from co-crystals to each of the nine hMcl-1 structures downloaded from the protein databank. The conformations of the docked structures were then compared to that in the X-ray crystal structure and RMSD values calculated (Table 3); the average RMSD values for the various ligands ranged from 0.879–1.390 Å with standard deviations from 0.116–0.331 Å. The structure 3WIX was selected as the pdb structure on which to proceed with further docking experiments as it accommodated co-crystal ligands to other pdbs with the lowest RMSD average. Through this approach, we also obtained structural information that helped to identify the active and passive amino acids in the cavity (mapped in Fig. 3 (b)) involved in the binding of all nine ligands to the 3WIX pdb. The superimposed ligands are shown in Fig. 3 (c)).
Table 2
MRC approach: Superposition of backbone of multiple hMcl-1 from various crystal structures and their calculated RMSD values (Å)
Proteins
|
4WMR
|
4ZBF
|
4ZBI
|
4OQ5
|
4OQ6
|
3WIX
|
3WIY
|
4HW2
|
Average
|
SD
|
Proteins
|
4WMR
|
|
|
|
|
|
|
|
|
0.888
|
0.174
|
4ZBF
|
0.86
|
|
|
|
|
|
|
|
0.644
|
0.156
|
4ZBI
|
0.68
|
0.43
|
|
|
|
|
|
|
0.638
|
0.134
|
4OQ5
|
0.66
|
0.83
|
0.73
|
|
|
|
|
|
0.858
|
0.143
|
4OQ6
|
1.06
|
0.78
|
0.85
|
0.90
|
|
|
|
|
0.873
|
0.102
|
3WIX
|
1.05
|
0.65
|
0.67
|
1.07
|
0.96
|
|
|
|
0.829
|
0.161
|
3WIY
|
0.72
|
0.46
|
0.46
|
0.71
|
0.70
|
0.74
|
|
|
0.658
|
0.117
|
4HW2
|
1.13
|
0.62
|
0.72
|
1.05
|
0.88
|
0.70
|
0.78
|
|
0.808
|
0.185
|
4HW3
|
0.94
|
0.52
|
0.56
|
0.91
|
0.85
|
0.79
|
0.69
|
0.58
|
0.730
|
0.155
|
Table 3
MLC approach: Docking of ligands and respective RMSD values (Å)
Proteins
|
4WMR
|
4ZBF
|
4ZBI
|
4OQ5
|
4OQ6
|
3WIX
|
3WIY
|
4HW2
|
4HW3
|
Ligands
|
4WMR
|
0.73
|
1.12
|
1.23
|
1.61
|
1.45
|
0.81
|
0.89
|
1.26
|
1.32
|
4ZBF
|
1.27
|
0.75
|
1.19
|
1.32
|
1.09
|
0.90
|
1.03
|
1.31
|
1.42
|
4ZBI
|
1.19
|
1.12
|
0.96
|
1.12
|
1.11
|
0.86
|
1.12
|
1.21
|
1.36
|
4OQ5
|
1.70
|
0.95
|
1.09
|
0.81
|
1.18
|
0.93
|
0.82
|
1.44
|
1.24
|
4OQ6
|
1.57
|
0.83
|
1.12
|
0.95
|
1.02
|
1.09
|
1.11
|
1.38
|
1.42
|
3WIX
|
1.20
|
0.98
|
1.03
|
0.99
|
1.08
|
0.69
|
0.78
|
1.25
|
1.07
|
3WIY
|
1.33
|
1.26
|
0.92
|
1.04
|
0.80
|
0.74
|
0.79
|
1.18
|
1.04
|
4HW2
|
1.87
|
1.14
|
1.30
|
1.61
|
1.49
|
0.98
|
0.86
|
1.12
|
1.13
|
4HW3
|
1.71
|
1.32
|
1.21
|
1.53
|
1.34
|
0.82
|
0.78
|
1.20
|
0.97
|
Average
|
1.397
|
1.052
|
1.117
|
1.220
|
1.173
|
0.869
|
0.909
|
1.261
|
1.219
|
Std. Dev
|
0.331
|
0.179
|
0.121
|
0.288
|
0.207
|
0.116
|
0.132
|
0.095
|
0.162
|
3.2 Evaluation of the precision and accuracy of docking methods
To evaluate both the precision and accuracy of docking methods, docking scores of a set of hMcl-1 inhibitors (Fig. 4) were obtained and compared with their corresponding Ki values [48]. These hMcl-1 inhibitors were chosen as they were prepared by the same group (Fesik) and the binding assay conditions were uniform for these inhibitors. Secondly, they all have been reported in co-crystal structures with hMcl-1, providing the experimental basis to validate docking methods. We employed three docking methods, with each ligand being docked into its own crystal structure (self-docking) and the results are summarized in Table 4 [12]. The self-docking [16] generally showed that the use of MOE [49] gave the lowest RMSD values for its docked poses when compared with crystal structure bound ligand conformation in hMcl-1, followed by AutoDock [50] and then VLifeDock [51].
Table 4
Comparison of scores from self-docking using various methods with hMcl-1 inhibitor Ki values.
pdb file
|
Ki value
(nM)
|
MOE
|
AutoDock
|
VLifeDock
|
References
|
Scorea
|
RMSDb
|
Scorea
|
RMSDb
|
Scorea
|
RMSDb
|
5FDO
|
361
|
-7.29
|
1.81
|
-8.30
|
1.94
|
-7.02
|
3.49
|
[45]
|
5FDR
|
0.94
|
-8.59
|
2.82
|
-7.22
|
3.07
|
-7.48
|
3.14
|
[45]
|
6BW2
|
21.0
|
-10.52
|
1.86
|
-9.94
|
2.30
|
-9.11
|
2.33
|
[47]
|
6BW8
|
< 1.00
|
-10.34
|
0.81
|
-9.84
|
1.36
|
-9.17
|
1.45
|
[47]
|
5IF4
|
< 1.00
|
-10.76
|
1.24
|
-10.14
|
1.36
|
-10.87
|
1.51
|
[46]
|
4HW2
|
55 ± 18
|
-8.41
|
1.12
|
-7.99
|
1.27
|
-8.02
|
1.30
|
[36]
|
4HW3
|
320 ± 10
|
-6.46
|
0.97
|
-6.47
|
1.19
|
-6.10
|
1.43
|
[36]
|
aScores in kJ/mol; bRMSD in Å |
To further assess the accuracy of the docking methods, iterative self-docking was performed using 3WIX [52] where the co-crystal ligand [7-(4-carboxyphenyl)-3-[3-(naphthalen-1-yloxy)propyl]pyrazolo[1,5-a]pyridine-2-carboxylic acid] of 3WIX was self-docked five times into its own binding site and each time it was compared with the orientations of its own conformation in the co-crystal structure by RMSD as shown in Table 5. The docking was commenced from random conformations of the ligand, and this, as well as affecting accuracy, can affect precision [53]. The precision of docking is also a concern in drug design, with precision defined by comparing RMSD values of poses obtained after repeated docking of the same ligand. The measurement of reproducibility of accuracy (NRMSE) was determined from the RMSD between the docked pose that is closest to the RMSD to that of the co-crystallized ligand of 3WIX as shown in Table 5. The "RMSE" value is the difference between the docked pose that has the highest RMSD value and the lowest RMSD value of docked pose and is thus a measure of the precision of the docking. These values are provided in Table 5 and the poses are shown in Fig. 5. The MOE triangle matcher docking method was the most accurate (NRMSE = 86%) compared to AutoDock (genetic algorithm, 55%) and VLifeDock (GRIP method-based docking: 36%). The MOE method was also the most precise as it had the lowest RMSE value. We also observed comparatively fast, accurate bound conformation prediction from VLifeDock when compared with AutoDock. However, AutoDock was more precise in generating docking poses [54] but less accurate in reproducing the co-crystal pose as compared to VLifeDock.
Table 5
Reproducibility of self-docking experiments performed five times of 3WIX and its co-crystal ligand*
Docking Phase Trials
|
RMSD (Å)
|
|
MOE
|
Autodock
|
VLifeDock
|
1
|
0.689
|
1.007
|
0.902
|
2
|
0.681
|
0.989
|
0.884
|
3
|
0.682
|
0.996
|
0.879
|
4
|
0.681
|
0.996
|
0.857
|
5
|
0.681
|
0.994
|
0.871
|
RMDSE (Δ = Å)
|
0.008
|
0.018
|
0.025
|
NRMSE
|
85.35
|
55.36
|
36.08
|
* Lower values for RMSE indicate higher precision. Lower values for RMSD indicate higher accuracy. RMSDE : Root-mean-square deviation error = RMSD (maximum value) – RMSD (Minimum value), lower RMSDE value indicates more reproducibility; Reproducibility was measure in terms of normalized root-mean-square deviation or error (NRMSD or NRMSE: This is fraction number which measures the degree of reproducibility) [55–57]. NRMSE = (Average RMSD attained / RMSDE), NRMSE value for any provided method tells the frequency of accuracy of getting docking conformation, higher values indicate better accuracy.
3.3 Docking of naturally occurring hMcl-1 inhibitors and related compounds
After evaluating the docking methods, screening of seven classes of naturally occurring hMcl-1 inhibitors was carried out; all these ligands had shown affinity in the low micromolar range.
3.3.1 Gymnochrome-F
To determine the binding conformation of Gymnochrome-F (1), we employed docking to 3WIX using MOE, AutoDock, and VlifeDock. Similar-pose ensemble clustering [60] confirmed the attained docking poses from these three different methods were all within RMSD 2 Å. This low RMSD value attained from these methods could be expected due to high rigidity possessed by the fused heteroaromatic rings in the ligand structure, which limits the number of binding conformations of Gymnochrome F. The docking of Gymnochrome-F showed its main interactions with His224 (π-π interaction, 3.81 Å) and H-bond donor and acceptor interaction with Asn260 (2.14 & 2.23 Å), as shown in Fig. 6.
3.3.2 Sponge-derived oxypolyhalogenated diphenyl ethers 2
In case of the diphenyl ether derivatives, we observed various bound conformations all with similar docking energies (within 0.2 kcal/mol). The smaller structure of these natural product derivatives enabled them to adopt a wide number of potential bound conformations in the larger active site of hMcl-1, as shown in Fig. 7. The structures were in proximity to Phe270 but with no interaction with for example Arg 263, that interacted with anacardic acid's salicylic acid residue, for example.
3.3.3. Anacardic acid derivatives (3a-g)
In case of anacardic acids, 3a showed close binding poses from all the three docking methods (1.17 Å RMSD). The docking predicted close interactions of polar phenolic head and carboxylic acid with Arg263 (2.36 & 2.21 Å) and intramolecular interaction (1.81 Å), as shown in Fig. 8.
The extra length in the hydrocarbon chain of 3b compared to 3a, projected the salicylic acid substructure of 3b more towards the NWGR domain of hMcl-1, where it interacted with Arg263 as a H-bond acceptor (2.0 & 2.14 Å), which also supports its improved hMcl-1 affinity (IC50 = 5.8 µM), compared to that of 3a (IC50 = 17.7 µM), as shown in Fig. 9.
In comparison to 3a and 3b, shortening of a hydrocarbon chain and incorporation of phenyl ring, as in 3c, further improved the affinity for hMcl-1 (IC50 = 0.6 µM) which could be understood with its additional π-π interaction with Phe270 which was missing in previously mentioned anacardic acid derivatives, as shown in Fig. 10. Clustering ensemble of docking conformations was found within RMSD of 1.67 Å and showed a π-π interaction with Phe270 (3.68 Å) along with Arg263 (2.14, 2.38 & 2.82 Å).
The docking of 3d, 3e, and 3f showed almost identical docking interactions with Arg263, as shown in Fig. 11, which is supported by their similar docking energies (-8.34, -8.47, and − 8.22 kcal/mol respectively). These anacardic acids' binding mode and binding energies are similar, which pointed out that the hydrocarbon chain length of (11–13 carbons) play a less significant role in influencing the binding of these molecules to hMcl-1.
In case of 3g (IC50 = 1.2 µM, ΔG = -8.00 kJ.mol− 1) when compared to 3e (IC50 = 0.2 µM, ΔG = -8.47 kJ.mol− 1) and 3f (IC50 = 0.2 µM, ΔG = -8.22 kJ.mol− 1), has a longer hydrocarbon chain which is also unsaturated which might led to reduced affinity which is most likely to be due to its steric effect (as shown in Fig. 12).
3.3.4 Endiandric Acid Analogues (4a-d)
The docking showed 4a (fused ring with carboxylic acid) binding conformation utilizes similar regions of hMcl-1 as 3c (salicylic acid derivative), which could possibly be based on similarity in their molecular structures (4a & 3c). However, structurally 4a has only a COOH group in its ring while 3c has both the acidic phenolic OH and COOH groups, where these polar functional groups were found to consistently interact with Arg263 of hMcl-1. The constrained fused ring in 4a might prevent its adoption of the optimal binding conformation for interaction with Arg263, and therefore could account for the observation of a weak H-bond interaction with Arg263 (3.51 Å) (, ΔG = -7.15 kJ.mol− 1) when compared to the 3c docking pose (has three H-bond acceptor interaction with Arg263: 2.14, 2.38 & 2.82 Å, ΔG = -7.98 kJ.mol− 1) see Fig. 10). Also, docking of 4a showed a π-π interaction with Phe270 (3.51 Å) of, as shown in Fig. 13.
Similar interactions were also seen with 4b (Ki = 13 µM), where it displayed an improved geometry for H-bonding as evaluated by its closer proximity to Arg263 (3.02 Å) and π-π interaction with Phe270 (3.50 Å) as shown in Fig. 14. The latter explains the marginal improvement in the in-vitro hMcl-1 affinity and docked binding energy, as indicated in Table 7.
The comparatively shorter analog in the endiandric acid class (4c) has an additional phenolic polar head and therefore shows an additional new H-bond donor interaction with peptide backbone of Leu267 and also has proximity with Phe270 (π-π interaction, 3.85 Å) when compared to longer analogs (4a and 4b), as shown in Fig. 15; therefore, explains why 4c achieved better in-vitro hMcl-1 affinity than later two, as shown in Table 7.
Ligand 4d has a different stereochemistry in its fused cyclic ring substructure with respect to the other analogs of this series, which leads to its COOH functionality being more inclined towards Arg263 (3.29 & 3.90 Å) as compared to a similar analog (like 4a), as shown in Fig. 16, which may account for its improved binding affinity (5.2 µM) over 4a (14.3 µM).
3.3.5 Marinopyrrole analogs
To study the interaction of marinopyrrole-A (maritoclax, 5a), Doi et al [22] utilized 1H-15N HSQC spectrum of 15N-labeled mMcl-1 data, and docking with mouse Mcl-1 (mMcl-1). Their study revealed some of the important molecular interactions with mMcl-1 protein: (a) the chloro group in one of the pyrrole ring of maritoclax was facing in towards the binding groove of mMcl-1, (b) the phenol group of the same pyrrole ring had a H-bond with Gly308, (c) the phenol group of another pyrrole group had a H-bond with Thr247, and (d) the carbonyl group had a H-bond with Asn204. Later, we utilized comparative sequence alignment of hMcl-1 with mMcl-1 to identify the corresponding amino acids of hMcl-1 with respect to those of mMcl-1 (as shown Table 6). Furthermore, our study revealed those amino acids of hMcl-1 which has preference to interact with marinopyrrole-A, with key species-specific differences: (a) identified two point mutations, G222-D241 and V262-I281; (b) hydrophobic character of the cavity of mMcl-1 than hMcl-1 (15 out of 25 in mMcl-1 & 13 out of 22 in hMcl-1 are hydrophobic amino acids) see Table 6.
Table 6
Comparison between mMcl and hMcl-1 active site residues. *
Corresponding amino acids
|
mMcl-1
|
hMcl-1
|
mMcl-1
|
hMcl-1
|
mMcl-1
|
hMcl-1
|
G150
|
-
|
N241
|
N260
|
R229
|
R248
|
I163
|
I182
|
G243
|
G262
|
F235
|
F254
|
T172
|
T191
|
I245
|
I264
|
D237
|
D256
|
G184
|
G203
|
S250
|
S269
|
R244
|
R263
|
G200
|
G219
|
F251
|
F270
|
V246
|
V265
|
Q202
|
Q221
|
V255
|
V274
|
|
|
R203
|
R222
|
V262
|
I281
|
|
|
N204
|
N223
|
V278
|
V297
|
|
|
R214
|
R233
|
L279
|
L298
|
|
|
L216
|
L235
|
F300
|
F319
|
|
|
N220
|
N239
|
Q306
|
|
|
|
G222
|
D241
|
G307
|
|
|
|
S228
|
S247
|
|
|
|
|
*The enlisted amino acids of mMcl-1 showed chemical shift perturbation (1H-15N HSQC spectrum) during the addition of Maritoclax (5a). The amino acids in black color were detected through changes in the 1H-15N HSQC spectrum of 15N-labeled mMcl-1. The unbolded and bolded amino acids of mMcl-1in black color showed 0.05 to 0.08 ppm chemical shift (δ) change respectively [61]. However, the amino acids shown in green were undetected due to large signal intensity change. Furthermore, we used sequence alignment to identify the corresponding amino acids of hMcl-1. Red colored amino acids in hMcl-1 representing the species-based point mutations among rat and human Mcl-1 proteins. |
Although, maritoclax (5a) showed similar bound conformation as reported for mMcl-1 in our docking studies with hMcl-1, but key amino acids residues interactions were found missing. To gain a better understanding, the hMcl-1 structure (PDB code: 3WIX) was superimposed with the mMcl-1 (PDB i.d. 2JM6) where RMSD value was found 6.91 Å. This high deviation provides the basis to explain differences observed in binding conformations of these proteins with maritoclax (5a). In our observation, Maritoclax (5a) is a dimer which contains two pyrroles: ring-A and ring-B (Fig. 2). The docking studies of maritoclax (5a) with hMcl-1 showed that the phenyl substituent on ring-A displays a π-π interaction with His224 and phenolic OH makes a H-bond donor interaction with Thr266 (1.51 Å) Fig. 17). Whereas the ring B showed an intramolecular H-bond interaction between NH-pyrrole and OH-Phenyl and also the carbonyl functionality showed two H-bond acceptor interactions with NH- functional group of guanidine moiety of Arg263 (2.06 & 2.64 Å). It has been observed that the maritoclax (5a) is in close proximity to Asp256, Met 231, Phe254, Leu 267, His224, Phe228, found in the cavity, which were also reported by Doi et al [22].
Later, Cheng et al. chemically modified maritoclax into cyclic derivatives which were evaluated for their Mcl-1 potencies. This work led to cyclic maritoclax (5b) with improved drug-like properties and a more potent hMcl-1 IC50 value (4.3 µM) compared to the parent maritoclax (8.9 µM) [62]. The correlation of improved drug-like properties and selective potency with cyclization of inhibitor is based on fact that, "in drug design, cyclization restrict the conformational flexibility of a ligand to improve its selectivity and potency for a given physiological target". However, docking evaluated herein failed to explain the differences in binding energies between maritoclax and its cyclic derivative (Fig. 18), which tells an intrinsic deficiency of docking algorithms to possess physicochemical parameter of ligand metrics.
3.3.6 MIM1 (Mcl-1 Inhibitor Molecule 1)
Similar to maritoclax (5), NMR-assisted docking was performed to identify the binding pose of MIM1 (6a) by Cohen et al [26]. The ensemble clustering of docked poses from MOE, AutoDock, and VlifeDock had RMSD values of 1.07 Å, 1.61 Å, and 2.02 Å respectively (see in Fig. 19). The attained docking pose in our study was compared with the previous reported binding of 6a, as was studied with hMcl-1 protein. In previous report, it had been reported that the 6a had a cyclohexyl group hydrophobic interaction with Val216 and Leu213 [26], which was not seen in our case as both residues were away from cyclohexyl functionality (> 5.5 Å), which is too long even for a weak hydrophobic interaction. However, other interactions were found similar to the previous study: (a) imidazole ring of His224 with a CH-π interaction (4.19 Å) with the methylated thiazole, (b) H-bond donor interaction (2.49 Å) with ortho-substituted –OH of pyrogallol functionality. On the other hand, the guanidine moiety of the side chain of Arg263 and alcoholic side chain (-OH) of Thr266 showed a H-bond acceptor interaction (2.47 and 2.06 Å) with para-substituted and ortho-substituted –OH of pyrogallol functionality. We also observed that the Asp256 is in close vicinity with Arg263 (2.63 Å), and could be better suited to contribute to binding the pyrogallol group via H-bonding [26].
3.3.7 Cryptosphaerolide
Cryptosphaerolide (7, TR-FRET, Ki = 11.4 µM) contains 2 substructures: cyclic (7a) and acyclic (7b). The absolute stereochemistry was reported for the cyclic portion but not for the acyclic substructure. Therefore, we generated all eight possible stereoisomers and docked them. These stereoisomers were later ranked based on their docking scores (Table 7). As shown in Fig. 20, docking of 7 showed that the cyclic substructure of all eight stereoisomers (coded in different colors in the figure) does not fit into the cavity and is projected out from the binding cavity; this explains why Fenical co-workers[27] did not observe any hMcl-1 inhibition activity for 7a.
3.3.8 Meiogynin derived hMcl-1 inhibitors (8a-c)
Meiogynin-A (8) derived compounds have shown a wide range of activity against antiapoptotic proteins of the Bcl-2 family [28]. Docking predicted similar binding interactions for meiogynins except in the case of their parent molecule 8 (Ki = 5.2 µM), which showed H-acceptor interactions with the imidazole side chain of His224. Meiogynin derivatives showed π-π interactions with Phe270. Previous docking studies of 8, which showed proximal binding with Arg100 and Tyr195, were also observed in our docking experiments (meiogynin A was found within 5 Å of these residues). However, the docking generated distinctive poses compared to those previously disclosed [63, 64] for meiogynins as shown in Fig. 21, 22, 23, and 24.
Table 7
Comparison of Ki's on hMcl-1 inhibition with their respective cumulative docking energies of naturally derived molecules.
Entry
|
Compound
|
hMcl-1
Ki/ IC50 in µM
|
Bcl-2 a/ Bcl-xL b
Ki/ IC50 = µM
|
Ref.
|
Triangle Matcher (kJ/mol)
|
AutoDock
(kJ/mol)
|
GRIP docking (kJ/mol)
|
RMSD
(Å)
|
1
|
1
|
3.3d
|
NR
|
[18]
|
-7.36
|
-7.71
|
-6.17
|
1.08
|
2
|
2a
|
2.4 ± 0.1
|
NR
|
[19]
|
-8.33
|
-8.29
|
-8.76
|
0.81
|
3
|
2b
|
8.9
|
NR
|
[19]
|
-8.42
|
-8.22
|
-8.67
|
0.72
|
4
|
2c
|
7.3
|
NR
|
[19]
|
-8.27
|
-8.16
|
-8.64
|
1.04
|
5
|
3a
|
17.7 ± 3.1 d
|
> 23 b, d
|
[20]
|
-7.14
|
-6.82
|
-7.17
|
1.22
|
6
|
3b
|
5.8 ± 0.3 d
|
3.2. ± 0.1 b, d
|
[20]
|
-7.83
|
-7.14
|
-7.67
|
2.09
|
7
|
3c
|
3.7 ± 2.0 d
|
16.3 ± 0.5 b, d
|
[20]
|
-7.98
|
-7.61
|
-8.13
|
1.58
|
8
|
3d
|
0.7 ± 0.1 d
|
1.2 ± 0.1 b, d
|
[20]
|
-8.34
|
-7.42
|
-8.91
|
2.18
|
9
|
3e
|
0.2 ± 0.1 d
|
0.3 ± 0.1 b, d
|
[20]
|
-8.47
|
-8.22
|
-9.09
|
1.97
|
10
|
3f
|
0.2 ± 0.1 d
|
0.2 ± 0.1 b, d
|
[20]
|
-8.22
|
-7.53
|
-8.76
|
2.05
|
11
|
3g
|
1.2 ± 0.9 d
|
5.7 ± 0.6 b, d
|
[20]
|
-8.00
|
-7.68
|
-8.27
|
2.31
|
12
|
4aa
|
14 ± 3.3 e
|
19.2 ± 1.6 b, e
|
[21]
|
-7.15
|
-6.71
|
-7.19
|
1.86
|
13
|
4b
|
13 ± 5.0 e
|
12.6 ± 0.2 b, e
|
[21]
|
-7.42
|
-6.32
|
-7.53
|
1.59
|
14
|
4c
|
5.2 ± 0.2 e
|
> 100 b, e
|
[21]
|
-8.02
|
-7.60
|
-8.11
|
0.88
|
15
|
4d
|
5.9 ± 0.5 e
|
19.4 ± 3.0 b, e
|
[21]
|
-7.89
|
-7.14
|
-8.02
|
1.03
|
17
|
5
|
8.9 ± 1.0 d
|
16.4 ± 3.3 b, d
|
[62]
|
-7.51
|
-6.67
|
-7.92
|
1.23
|
18
|
5a
|
4.3 ± 1.5 d
|
3.4 ± 0.9 b, d
|
[62]
|
-7.06
|
-6.39
|
-7.84
|
1.70
|
19
|
6a
|
4.72 d
|
NR
|
[26]
|
-8.77
|
-7.88
|
-8.90
|
2.13
|
20
|
7 (R, R, R)
|
NA c
|
NR
|
[27]
|
-6.88
|
-5.29
|
-6.16
|
2.64
|
21
|
7 (R, R, S)
|
NA c
|
NR
|
[27]
|
-7.17
|
-6.02
|
-6.31
|
2.37
|
22
|
7 (R, S, S)
|
NA c
|
NR
|
[27]
|
-7.04
|
-5.89
|
-6.20
|
3.14
|
23
|
7 (S, S, S)
|
NA c
|
NR
|
[27]
|
-7.22
|
-6.17
|
-6.47
|
1.76
|
24
|
7 (S, R, R)
|
NA c
|
NR
|
[27]
|
-6.53
|
-5.56
|
-5.90
|
2.22
|
25
|
7 (S, S, R)
|
NA c
|
NR
|
[27]
|
-6.70
|
-5.82
|
-6.13
|
2.90
|
26
|
7 (R, S, R)
|
NA c
|
NR
|
[27]
|
-7.34
|
-6.31
|
-6.41
|
2.46
|
27
|
7 (S, R, S)
|
NA c
|
NR
|
[27]
|
-7.12
|
-6.12
|
-6.28
|
1.94
|
29
|
8
|
5.2 ± 1.2 e
|
1.46 ± 0.12 a, e / 8.30 ± 1.20 b, e
|
[28]
|
-6.66
|
-6.42
|
-6.24
|
1.48
|
30
|
8a
|
0.46 ± 0.06 e
|
0.83 ± 0.16 a, e / 2.19 ± 0.09 b, e
|
[28]
|
-7.22
|
-7.14
|
-6.94
|
1.14
|
31
|
8b
|
5.92 ± 0.47 e
|
> 23 a, e/ 8.48 ± 0.40 b, e
|
[28]
|
-7.33
|
-7.25
|
-7.22
|
1.85
|
32
|
8c
|
0.56 ± 0.04 e
|
1.54 ± 0.44 a, e / 2.44 ± 0.02 b, e
|
[28]
|
-6.97
|
-6.89
|
-6.77
|
1.57
|
a Bcl-2 protein inhibition; b Bcl-xL protein inhibition; NA c isolated cryptosphaerolide (7) had a Ki of 11.4 µM so all isomers were listed here: d IC50 values; e Ki values; NR not reported. |