Structure based virtual screening and hit identification of purine compounds:
High-throughput virtual screening utilizing molecular docking was conducted on an entire library of purine-type compounds, aiming at the ATP binding pocket of katanin. The objective was to pinpoint purine compounds exhibiting a stronger binding affinity, employing AutoDock Vina 34. Within this screening process, we scrutinized 2,76,280 purine-type compounds, examining their interaction specifically with ATP binding site of katanin, as depicted in Fig. 2. After the initial screening, compounds were sieved based on their binding affinity, culminating in the selection of the top 5 hit compounds exhibiting the lowest binding energy greater than ≤ -10 kcal/mol with katanin, as outlined in Table 1. This selection process was executed through InstaDock 35.
The notable binding affinity exhibited by these top 5 compounds led us to investigate their potential for the development of drugs. To assess viability of selected compounds, we conducted physicochemical, toxicity, and biological characteristics using ADME-T and pass prediction methodologies.
PASS prediction and ADME-T properties:
The selected top 5 compounds (PubChem IDs: 123629569, 163388234, 122589735, 163555323, 156185498) were underwent for pass prediction to check its biological activity prediction by using Way2drug web server 36. The selected purine rich compound from the PubChem database along with their PubChem ID, and 2D structures are mentioned in Table 1. Here, all the compounds exhibited Pa values below 0.3, Pa values greater than Pi values, suggesting their active nature and listed in Table 2. Moreover, all these compounds demonstrated anti-neoplastic and anti-metastatic activity, and anti-cancer activity.
Table 2
Biological activity of the selected hit compounds using the Way2drug webserver.
S.No.
|
PubChem
Compound ID
|
Pa
|
Pi
|
Properties
|
1
|
123629569
|
0.213
|
0.117
|
Proto-oncogene tyrosine-protein kinase Fgr inhibitor
|
0.140
|
0.102
|
Liver fibrosis treatment
|
0.254
|
0.092
|
Cystic fibrosis treatment
|
2
|
163388234
|
0.315
|
0.038
|
Prostate cancer treatment
|
0.438
|
0.091
|
Antineoplastic: sarcoma, lymphoma
|
3
|
122589735
|
0.302
|
0.222
|
Antineoplastic (non-Hodgkin's lymphoma)
|
0.185
|
0.183
|
Antimetastatic
|
0.184
|
0.084
|
Prostate cancer treatment
|
0.258
|
0.044
|
Antineoplastic enhancer
|
4
|
163555323
|
0.552
|
0.056
|
Antineoplastic
|
5
|
156185498
|
0.489
|
0.005
|
Antineoplastic enhancer
|
0.411
|
0.020
|
Prostate cancer treatment
|
0.263
|
0.049
|
Antineoplastic alkaloid
|
0.123
|
0.076
|
Antineoplastic: lymphocytic leukemia, bladder cancer, glioblastoma multiforme, lymphoma, glioma
|
The top five compounds also underwent pharmacokinetic as well as toxicology predictions using SWISS-ADME 37 and pkCSM webserver 38, respectively. All the compounds molecular weight was > 400kDa that indicates stability. Four compounds show greater lipophilicity that indicated by a larger logP value, which implies that the substance has a stronger propensity to diffuse into lipid-rich environments like cell membranes. Water solubility criteria were categorized as insoluble (<-10), poorly soluble (-6), moderately (-4), soluble (-2), and very soluble (0), with Esol indicating poor solubility for 123629569 and 156185498 and high solubility for 122589735. Lipinski's rule, specifying < 5 hydrogen bond donors, < 10 hydrogen bond acceptors, and molecular weight < 500 Da, showed only one violation, which is acceptable. Bioavailability was predicted as 0.55, indicating neutrality. None of the compounds exhibited PAINS (Pan-Assay Interference Compounds). Synthetic availability ranged between 1 (very easy) to 10 (difficult), with all compounds scoring < 5 as shown in Table 3. After analysing the top 5 compounds concerning their pass prediction and ADME-T properties, we selected top 3 compounds 123629569, 163388234, and 122589735 for further molecular docking study.
Table 3
ADME-T properties of the selected top five compounds
ADME Parameters
|
Properties
|
123629569
|
163388234
|
122589735
|
163555323
|
156185498
|
Physiochemical Properties
|
Formula
|
C26H25F3N6O2
|
C67H44F3N12O
|
C24H33N9O3
|
C44H28FN5
|
C39H25N5O
|
Molecular weight
(g/mol)
|
510.51
|
1090.14
|
495.58
|
645.73
|
579.65
|
Absorption
|
GI
Absorption
|
Low
|
High
|
High
|
Low
|
Low
|
Water
Solubility
|
Poorly soluble
|
Insoluble
|
Soluble
|
Insoluble
|
Poorly soluble
|
Distribution
|
BBB
Permeation
|
No
|
No
|
No
|
No
|
No
|
Lipophilicity (ILogP)
|
3.02
|
0
|
2.79
|
4.97
|
4.84
|
Metabolism
|
CYP2D6 Substrate/ Inhibitor
|
Yes
|
No
|
Yes
|
No
|
No
|
Excretion
|
OCT2
Substrate
|
No
|
Yes
|
No
|
Yes
|
Yes
|
Toxicity
|
AMES toxicity
|
No
|
Yes
|
No
|
Yes
|
Yes
|
Maximum Tolerance Dose
|
0.472
|
0.438
|
0.644
|
0.438
|
0.438
|
Hepatotoxicity
|
Yes
|
No
|
Yes
|
No
|
No
|
Skin Sensitisation
|
No
|
No
|
No
|
No
|
No
|
Drug likeness and medicinal chemistry
|
Lipinski
|
1
|
3
|
1
|
2
|
2
|
Bioavailability score
|
0.56
|
0.17
|
0.55
|
0.17
|
0.17
|
PAINS
|
0
|
0
|
0
|
0
|
0
|
Synthetic accessibility
|
4.15
|
7.39
|
4.81
|
4.22
|
4.09
|
Interaction of katanin with purine type compounds using docking:
First, we performed a control docking of katanin with ATP using AutoDock 4.2.7 39, and the minimum energy docked conformation of ATP was found to be -4.86 kcal/mol (Fig. 3B and Table 4). The analysis of katanin-ATP complex shows that the ATP is stabilized by the bonded and non-bonded type of interactions as shown in Fig. 3 and Table 4. ATP forms a conventional hydrogen bonding interaction with residues Gly252 (1.68 and 2.33Å), Thr253 (2.21 Å), Gly254 (2.81 and 2.10 Å), Lys255 (2.52 Å), Thr256 (1.71 and 3.05 Å), Leu257 (2.57 Å), Asp210 (2.41 Å), Thr422 (2.04Å), whereas Leu257 forms a π-sigma, and Leu390 forms π-alkyl type of non-bonded interactions as shown in Fig. 3A, 3B and Table 4.
Similarly, molecular docking was performed to explore the binding mode and affinity of selected compounds 122589735, 123629569 and 163388234 with katanin using AutoDock 4.2.7 39. The least binding energy conformation of compounds 122589735, 123629569 and 163388234 were found to be -8.57, -8.85, and − 8.51 kcal/mol, respectively as shown in Fig. 3 and Table 4.
Notably, compound 122589735 exhibited the lowest binding energy with katanin compared to all other compounds analysed. These findings highlight the considerable potential of these compounds for katanin binding, particularly targeting the ATP site. Therefore, to delve into the mechanisms of bonded and non-bonded interactions with katanin, we conducted additional analyses of docked complexes and thoroughly discuss our findings.
Table 4
Analysis of 2D interactions of drug compounds with Katanin receptor after molecular docking.
PubChem Compound Id
|
Binding Energy
(kcal/mol)
|
Atoms involved in binding
|
Bond type
|
Distance
|
Angle
|
Fig
|
Katanin-ATP
|
-4.86
|
GLY252:HN - ATP501:O20
THR253:HN - ATP 501:O1B
GLY254:HN - ATP 501:O5'
GLY254:HN - ATP 501:O1B
LYS255:HN - ATP 501:O1B
THR256:HN - ATP 501:O3A
LEU257:HN - ATP 501:O2A
ATP 501:H61 - ASP210:O
GLY252:CA - ATP 501:O2G
THR256:CB - ATP 501:O1A
THR422:HG1 - ATP501
|
H Bond
H Bond
H Bond
H Bond
H Bond
H Bond
H Bond
H Bond
H Bond
H Bond
H Bond
|
1.68
2.21
2.81
2.10
2.52
3.05
2.57
2.41
2.33
1.71
2.04
|
164.55
119.63
121.04
143.09
133.19
99.24
127.69
143.99
126.59
154.75
107.42
|
3B
|
Katanin − 122589735
|
-8.85
|
ASN360:ND2 - Drug: O
Drug: H - ALA212:O
Drug: C - THR422:OG1
Drug: C - ASP308:OD2
Drug: C - THR253:O
|
H Bond
H Bond
CH Bond
CH Bond
CH Bond
|
3.15
2.50
3.16
3.30
3.38
|
93.24
-
90.31
125.53
94.84
|
3C
|
Katanin − 123629569
|
-8.57
|
GLY252:CA - Drug:O
GLY418:CA - Drug: N
Drug: C - GLY418:O
Drug: C - THR422:OG1
|
CH Bond
CH Bond
CH Bond
CH Bond
|
3.66
3.22
3.50
3.19
|
93.75
105.9
96.9
113.2
|
3D
|
ϯKatanin- 163388234
|
-8.51
|
-
|
-
|
-
|
-
|
3E
|
ϯ There are no conventional and CH-type hydrogen bonding interactions with katanin.
The analysis of the katanin-122589735 complex (Fig. 3) shows that stability of the 122589735 compound is attributed to conventional hydrogen bonding interactions with residues Asn360 (3.15 Å), Ala212 (2.50 Å), whereas CH bonding interactions with residues Thr422 (3.16 Å), Asp308 (3.30 Å), and Thr253 (3.38 Å) as shown in Table 4 and Fig. 3C. In addition, 122589735 makes an alkyl interaction with Lys255 (5.25 Å and 4.84 Å), Leu257 (5.28 Å), Ala358 (4.32 Å), Pro251 (4.66 Å), and π-Alkyl interactions with Leu257 (4.66 Å), Leu390 (5.47 Å), Ala212 (5.22 Å), Pro382 (4.36 Å), and Leu390 (4.35 Å) as shown in Table 4 and Fig. 3C.
Further analysis of katanin-123629569 complex (Fig. 3) shows that the 123629569 compound is stabilized due to CH bonding interactions with residues Gly252 (3.66 Å), Gly418 (3.22 Å and 3.50 Å), and Thr422 (3.19 Å), also the presence of Halogen (fluorine) type interaction was seen for Asp210 (3.42 Å). In addition, 123629569 compound forms π-Sigma interactions with Leu257 (3.62 Å), Leu390 (3.82 Å) and Ala419 (3.84 Å) as shown in Table 4 and Fig. 3D. Also, Amide-π stacked bonds with Thr253 (4.47 Å), Gly418 (4.24 Å) and Gly418 (5 Å) (Table 4 and Fig. 3D). Moreover, Alkyl type of interactions were contributed to Ala419 (4.16 Å), Pro382 (3.52 Å), Leu390 (4.26 Å), Leu257 (5.16 Å), and π-Alkyl bonds for Leu257 (5 Å) as well as Ala419 (4.63 Å) as shown in Table 4 and Fig. 3D. Next, the analysis of the katanin-163388234 complex (Fig. 3) shows that the 163388234 compound forms π-Donor hydrogen bond for Thr256 (3.98 Å and 3.74 Å), and Asn272 (3.69 Å) (Table 4 and Fig. 3E), π-Sigma bond with Ile393 (3.82 Å) and π-Alkyl bonds for Leu257 (4.56 Å), Val206 (5.25 Å), Leu257 (5.24 Å and 4.55 Å) as well as Lys255 (5.32 Å) as shown in Table 4 and Fig. 3E.
The analysis of docking results revealed that katanin with 122589735 complex is stabilized by both bonded and non-bonded types of interactions. However, compound 163388234 forms only non-bonded type of interactions with katanin, as it lacks electron bond donor groups such as O, N, S, etc. In addition, the compound 163388234 shows lower binding affinity with katanin as shown in Table 4 and Fig. 3E. Hence, to explore the refined binding mode and affinity of katanin with ATP, 122589735 and 123629569 compounds, molecular dynamics simulations were employed.
Molecular dynamics (MD) simulation
MD simulations were performed to investigate the interaction of katanin with ATP, 122589735 and 123629569 using Gromacs 2021.5 52. The least energy docked conformation of katanin, katanin-ATP, katanin-123629569 and katanin-122589735 shown in (Fig. 3), were considered as starting conformation for MD simulation. The simulations were performed for 500 ns, to obtain detailed conformational and structural changes in the katanin (see supplementary movies 1–4). The stability of the simulation systems were assessed by plotting the root mean square deviation (RMSD) of the Cα backbone atoms of protein (Fig. 4A). RMSD plot revealed that all the simulation systems reached their equilibrium after 300ns (Fig. 4A). Overall, katanin with ATP and drug complexes show lower RMSD value compared to the katanin in apo form (Fig. 4A), this shows that the ATP and drug compound has profound effect on structure and dynamics of katanin (see supplementary movie 1–4). The katanin-123629569 as it shows the higher fluctuations compared to all other katanin complexes after 300ns (Fig. 4A). To gain more insight on the impact of drug binding and conformational changes in the katanin structure, root mean square fluctuations (RMSF) of Cα atoms were performed (Fig. 4B).
It is revealed that katanin bound to ATP and 122589735 compounds showed a lower fluctuations (Fig. 4B) compared to the katanin and katanin-123629569. The ATP binding site residues of katanin (141 to 171) shows reduced fluctuations of katanin 122589735 compared to the katanin alone and katanin with ATP and 123629569 (Fig. 4B). Moreover, katanin-122589735 complex revealed lower conformational changes in 122589735 compound (Supplementary Movie 3) while 123629569 compound show higher conformational fluctuations (Supplementary Movie 4). This might be because compound 122589735 forms two hydrogen bonds with Asn360 and Ala212, along with three CH bonds with Thr422, Asp308, and Thr253 of katanin (Fig. 4B-4C and Table 4). In contrast, compound 123629569 only engages in non-bonded interactions. Altogether, katanin with compound 122589735 show a profound effect on the structure and dynamics of katanin.
To further check the compactness of the protein, radius of gyration (Rg) (Fig. 4C) and solvent-accessible surface area (SASA) (Fig. 4D) were calculated. The Rg plot analysis revealed that katanin-ATP and katanin-122589735 complexes exhibit a lower Rg value compared to katanin and katanin-123629569 complexes (Fig. 4C). This suggests that katanin adopts a more compact conformation when bound to ATP and compound 122589735 (Fig. 4C). Similarly, the SASA plot complements Rg analysis by focusing on the surface area of the protein accessible to the solvent molecule. The collective SASA values of all the systems ranged between 160-180nm2 (Fig. 4D). To comprehensively characterize the protein's conformational changes upon binding to ATP and drug compounds throughout the simulation, we employed a multi-pronged approach which includes principal component analysis (PCA), free energy landscape, hydrogen bonding interaction, and binding energy calculations.
Principle component analysis (PCA):
PCA was carried out using the gmx_covar and gmx_anaeig modules of Gromacs 2021.5 to understand the essential motions of the katanin, katanin-ATP and katanin-122589735, and katanin-123629569 complexes. Here, the eigenvectors, known as PCs (principal components) were investigated as shown in Fig. 4E. PCA analysis revealed that there was a higher diversity of conformations when katanin was in an unbound state during the simulation while, for the katanin-ATP, katanin-122589735 and katanin-123629569 complexes showed lower conformational diversity as shown in Fig. 4E. PCA analysis showed the highest diversity of conformations for katanin during the simulation (PCA2:7, PCA1:7) compared to other complexes. As evidenced by the PCA scores, the katanin-122589735 complex scored (PCA2: 4, PCA1: 8) and the katanin-123629569 complex scored (PCA2: 4, PCA1: 10). However, the katanin-ATP showed the least diversity in the conformations. Overall, katanin in its unbound state has higher variations in the conformations over the time of 500 ns but, upon drug binding the dynamics of katanin are affected thus leading to a stable state. To gain a deeper understanding of the energetics involved, we employed free energy landscape analysis to visualize the various energetic states of katanin, katanin bound to ATP, and katanin in complex with the drug compounds.
Free energy landscape (FEL)
FEL analysis shows the protein conformational space concerning energy and time 53. This allowed us to visualize the most stable conformations and identify potential energy barriers for transitions between varying states. For all the landscapes (Fig. 5A-5H), the initial minimum represents the filtering of the most stable conformation and has the lowest energy with a conical termination. As observed in the MD analysis results and PCA, katanin in unbound nature displays large conformation states and takes time to achieve the least energy conformation (Fig. 5A and 5B), similarly, a greater number of high energy states are observed as seen in the contour map plot of katanin (Fig. 5B). The scenario changes when the katanin is bound to ATP, and stabilizes the katanin as observed in the energy funnel with confined basin (Fig. 5C and 5D).
Similar observations are made concerning katanin-drug complexes. Wherein, katanin-122589735 complex had the least energy range between 0-1.62 kJ/mol for the deep energy minima (Fig. 5E and 5F). The katanin-123629569 shows single minima but undergoes more variations/transition state in the structural folding as compared to katanin-122589735 to achieve the least energy state (Fig. 5G and 5H). These results infer that the katanin-122589735 complex achieves the global state conformation sooner than the katanin-123629569 complex (Fig. 5E-5H). To gain a deeper understanding of the interactions between katanin and drug compounds, we specifically investigated hydrogen bonding through a dedicated analysis.
Hydrogen bond interaction analysis
This analysis quantified and characterized the hydrogen bond formation between katanin and ATP, 122589735 and 123629569 compounds during the 500 ns time steps (Supplementary Fig. 1). Katanin-ATP complex shows more stable and frequent hydrogen bond formation (Supplementary Fig. 1A). Further, hydrogen bond analysis shows that the katanin-122589735 complex (Supplementary Fig. 1B) forms stable hydrogen bonds as compared to the katanin-123629569 complex (Supplementary Fig. 1C). Whereas the number of hydrogen bonds increases after 300ns for the katanin-122589735 complex indicating stronger interaction between the drug and katanin (Supplementary Fig. 1A). The hydrogen bond analysis shows that 122589735 is stable at the ATP binding pocket of katanin and from constant the number of hydrogen bonds is over time (Supplementary Fig. 1B). Hence, to investigate the binding affinity of drug compounds, we employed the binding energy calculations and residue per decomposition analysis using the gmx_MMPBSA tool 49.
Binding energy calculations
Table 5
Binding energy calculation of katanin with ATP and drug compounds. All energies are in kcal/mol.
Katanin-drug complex
|
ΔEvdw
|
ΔEele
|
ΔEgas
|
ΔEsol
|
ΔEbind
|
Katanin-ATP
|
-40.74
|
215.51
|
174.76
|
-197.46
|
-22.70
|
Katanin-122589735
|
-45.45
|
-20.63
|
-66.08
|
32.49
|
-33.58
|
Katanin-123629569
|
-39.20
|
-13.73
|
-52.93
|
25.39
|
-27.54
|
To investigate the binding affinity of katanin with ATP and drug compounds, we employed binding energy calculations using the MM-GBSA method through gmx_MMPBSA tool 49. The equilibrated last 100 ns (a total of 5000 frames) were considered for the binding energy calculations. The energy data analysis revealed that katanin had a higher binding affinity with compound 122589735 compared to ATP and compound 123629569 as shown in Table 5. The order of binding affinity decreases in the order of 122589735 (-33.58 kcal/mol) > 123629569 (-27.54 kcal/mol) > ATP (-22.70 kcal/mol). Furthermore, the analysis revealed that the Van der Waals and electrostatic interactions play a significant role in the binding of the 122589735 compounds with katanin whereas, the loss of these interactions reduces the affinity of 123629569 for katanin. To further investigate the contribution of binding site residues, we performed per-residue energy decomposition analysis. Additionally, the per-residue contributions of katanin interacting with the drug and influencing binding affinity were determined using decomposition analysis with the gmx_MMPBSA tool (Supplementary Fig. 2).
The residues decomposition analysis of katanin-ATP complex shows that Ile28, Leu31, Thr70, Gly71, Leu74, Gly235, Ala236, Ile238, and Thr239 are involved in the binding with ATP (Supplementary Fig. 2A). Whereas katanin-122589735 complex had a relatively higher number of active residues contributing to energy than the other complexes (Supplementary Fig. 2B). Notably, Leu74, Ala236, and Thr239 exhibited the highest energy contributions. In katanin-123629569 complex, Gly71, Gly235, and Thr239 were found to have significant roles in drug binding (Supplementary Fig. 2C). Overall, residue decomposition analysis suggests that residues in the ATP binding site, such as Thr70, Gly71, Leu74, Gly235, Ala236, and Thr239, are common active site residues of katanin that interact with both ATP and the drugs (Supplementary Figs. 2A-2C).