In silico evaluation of apoptogenic potential and toxicological profile of triterpenoids.

AIM: Caspases-3 and 8 are key mediators of intrinsic and extrinsic pathway of apoptosis, respectively. Triterpenoids of natural and synthetic origin reported as anticancer agents with apoptotic potential and hence may prove to be good candidates for in silico testing against caspases-3 and 8.
MATERIALS AND METHODS: Various naturally-occurring and synthetic triterpenoids were subjected to activity prediction using PASS Online software, and among them, 67 compounds were selected for further processing. Protein structure of caspase-3 (3DEI) and caspase-8 (3KJQ) was obtained from the protein data bank and docked with selected triterpenoids using AutoDock Tools and AutoDock Vina. Toxicological profile was predicted based on clinical manifestations using PASS online software.
RESULTS: The high docking score of -10.0, -9.9, -9.8, and -9.5 were shown by friedelin, tingenone, albiziasaponin A, and albiziasaponin C, respectively, for caspase-3, and -11.0, -9.6, -9.6, and -9.4 by β-boswellic acid, bryonolic acid, canophyllic acid, and CDDO, respectively, for caspase-8. Possible adverse events were predicted with varying degree of probability and major relevant effects were reported. Hydrostatic interactions along with formation of hydrogen bonds with specific amino acids in the binding pocket were identified with each triterpenoid.
CONCLUSION: Lead molecules identified through this in silico study such as friedelin, tingenone, albiziasaponin, bryonolic acid, and canophyllic acid may be utilized for further in vitro/in vivo studies as apoptotic agents targeting caspases-3 and 8.

Introduction

A poptosis is a regulatory mechanism controlling cell proliferation in physiologic and pathologic conditions.[12] It plays a vital role in development, aging, and defense mechanism through the immune system. In oncology, apoptosis proved to be the prime target for anticancer research which comprises two pathways: intrinsic mitochondrial-dependent pathway and the extrinsic death receptor-mediated pathway. Caspases, cysteine-dependent aspartate-directed proteases, are the vital enzymes which synchronize the cellular events of apoptosis. Caspase-8, a member of initiator caspases, mediates extrinsic pathway,[3] while caspase-3, a member of execution caspases, mediates intrinsic pathway.[4]

Triterpenoid saponins are the phytoconstituents reported to exhibit antimetastasis, antiproliferative, anti-angiogenic, and reversal of multidrug resistance effects through induction of apoptosis and help in cell differentiation.[5] Saponins were reported to inhibit the growth of human breast cancer cell lines and induce apoptosis in Jurkat cells.[6] Various triterpenoids such as gymnemic acid,[78] rotundic acid,[9] and euscaphic acid[10] are either evaluated or suggested to be a promoter of caspase-3- or caspase-8-mediated apoptosis.

This proposed apoptogenic potential of various triterpenoids motivated us to perform in silico screening of naturally occurring and synthetic triterpenoids. We used combination of pharmacological activity and toxicity prediction softwares such as “PASS online”[111213] along with molecular docking software “Autodock Vina”[14] and “LigPlot+”[15] which generate schematic two-dimensional (2D) representations of protein-ligand complexes. PASS online is an online activity prediction software which claims 95% accuracy in their results. It predicts the activity by comparing the provided structure with over 260,000 of drug-like biologically active compounds.[16] AutoDock Vina, one of the most widely used docking softwares, based on gradient optimization algorithm uses empirical scoring function to achieve speed and accuracy in predicting binding modes for ligand on protein molecules,[14]” LigPlot+,” which generates 2D representations of protein-ligand complexes, was used to identify amino acids in target protein where ligand binds, and nature of binding in terms of “type of bond,” i.e., hydrogen bond, covalent bond, etc.[15]

Materials and Methods

Pharmacological activity prediction in PASS Online

Various naturally-occurring and synthetic triterpenoids were identified based on the literature search using PubMed and Google Scholar search engines. Structures of these triterpenoids were either downloaded in molecular data file format (MDL Molfile), i.e., mol extension from websites https://pubchem.ncbi.nlm.nih.gov/, http://www.chemspider.com/, and http://www.ebi.ac.uk/ or downloaded in available format and converted to.mol extension using OpenBabel 2.4.1. Activity prediction is carried out with PASS online as reported elsewhere. Structures in.mol file format were uploaded in PASS online for prediction. Predicted activities were filtered by searching results for caspase-3 and caspase-8 and 67 compounds were selected for further processing. Corresponding Pa (probability to be active) and Pi (probability to be inactive) values were recorded.

Molecular docking with AutoDock Vina

The MDL Mol files of selected triterpenoid structures were converted to protein data bank (PDB) file format (.pdb format) using OpenBabel 2.4.1 and processed with AutoDock Tools 4.2.6. Protein structures, i.e., caspase-3 (PDB ID-3DEI) and caspase-8 (PDB ID-3KJQ) were obtained from Research Collaboratory for Structural Bioinformatics website in.pdb format.

Preparation of ligands for docking

The 3D.mol format structures of triterpenoids were converted to PDB file format using Open Babel 2.4.1 and accessed through AutoDock Tool. Polar hydrogens were added. Gasteiger charges were computed, nonpolar hydrogens were merged, torsion was defined, and files were saved as.pdbqt format.

Preparation of proteins for docking

Caspase-3 (PDB ID-3DEI) and caspase-8 (PDB ID-3KJQ) files accessed using AutoDock Tool. The attached ligands were identified and deleted before processing. Water molecules were removed; both polar and nonpolar hydrogens were added. Gasteiger charges were computed and files were saved as.pdbqt format.

Ligand binding sites on protein

Ligand-binding sites on protein, i.e., caspase-3 and caspase-8 were identified using MetaPocket 2.0, a meta-server which uses consensus method to predict the binding sites using eight methods: LIGSITEcs, PASS, Q-SiteFinder, SURFNET, Fpocket, GHECOM, ConCavity, and POCASA. The best three pocket sites were identified and used for docking with triterpenoid structures.

Determination of grid box and molecular docking

Parameters for grid box were decided based on pocket identified by MetaPocket 2.0. These values were used to dock the corresponding protein with triterpenoid structures in AutoDock Vina. Protein-ligand binding was visualized using UCSF Chimera and 2D schematic diagrams of interactions were obtained with LigPlot+ software. Amino acids showing interactions with ligand structures were identified.

Toxicity profile

Major adverse effects were predicted depending on their structural peculiarities by comparing with the data of over 260,000 compounds using PASS prediction software. General Unrestricted Structure-Activity Relationships (GUSAR) software is used to predict LD50 values in rats by various routes of drug administration.

Results

The PASS online predictions related asiaticoside, madecassoside, maniladiol, betulinic acid, oleanolic acid, hederagenin, and crotalic acid have highest probability to be active (Pa) for stimulation of caspase-3. The Pa scores for these triterpenoids were 0.990, 0.988, 0.987, 0.985, 0.984, 0.982, and 0.956, respectively. While albiziasaponin C, phytolaccagenin, arjunolic acid, hyptatic acid, coussaric acid, and glycyrrhizin showed Pa values 0.975, 0.921, 0.909, 0.909, 0.893, and 0.873, respectively, for stimulation of caspase-8. Table 1 denotes Pa and Pi values of triterpenoids for caspase-3 and caspase-8.

Table 1

PASS online prediction and molecular docking studies of triterpenoids

Serial number	Name of triterpenoids	Structure	Caspase-3 stimulation					Caspase-8 stimulation					Previously reported
			PASS online prediction data		Binding energy (Kcal/mol)	Amino acid residue	Hydrogen bond	PASS online prediction data		Binding energy (Kcal/mol)	Amino acid residue	Hydrogen bond
			Pa	Pi				Pa	Pi
1	3epi Katonic acid[17]		0.916	0.003	−8.6	Glu123	1	0.841	0.001	−7.8	Thr149	11
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						His121					His317
						Tyr204
2	23 hydroxyursolic acid[18]		0.896	0.004	−8.7	Glu123	1	0.839	0.001	−5.8	Thr149	11	Activation of caspase- 3 and caspase-8[19]
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						His121					His317
						Tyr204
3	Albiziasaponin A[20]		0.987	0.001	−9.4	Glu123	1	0.969	0.000	−8.9	Thr149	11
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						His121					His317
						Tyr204
4	Albiziasaponin B[20]		0.988	0.001	−9.3	Glu123	1	0.975	0.001	−8.2	Thr149	11
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						His121					His317
						Tyr204
5	Albiziasaponin C[20]		0.988	0.001	−9.8	Glu123	1	0.975	0.000	−8.2	Thr149	11
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						His121					His317
						Tyr204
6	Albiziasaponin D[21]		0.940	0.003	−9.2	Glu123	1	0.925	0.001	−8.0	Thr149	11
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						His121					His317
						Tyr204
7	Albiziasaponin E[21]		0.965	0.002	−9.9	Glu123	1	0.935	0.000	−8.2	Thr149	11
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						His121					His317
						Tyr204
8	Alpha- boswellic acid[17]		0.907	0.003	−8.8	Glu123	1	0.841	0.001	−7.4	Thr149	11
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						His121					His317
						Tyr204
9	Arjunolic acid[18]		0.976	0.002	−8.6	Glu123	1	0.909	0.001	−7.4	Thr149	11	Activation of caspase-3[22]
											Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						His121					His317
						Tyr204
10	Asiatic acid[18]		0.865	0.004	−8.7	Glu123	1	0.829	0.001	−7.1	Thr149	11	Activation of caspase-3[23]
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						His121					His317
						Tyr204
11	Asiaticoside[18]		0.990	0.001	−8.5	Glu123	1	0.968	0.000	−7.2	Thr149	11	Activation of caspase-3[24]
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						His121					His317
						Tyr204
12	Barrigenic acid[18]		0.353	0.074	−4.0	Glu123	1	0.525	0.013	−3.2	Thr149	11
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						His121					His317
						Tyr204
13	Bartogenic acid[18]		0.813	0.005	−7.3	Glu123	1	0.792	0.002	−6.1	Thr149	11
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						His121					His317
						Tyr204
14	B-boswellic acid[17]		0.724	0.010	−9.3	Glu123	1	0.744	0.002	−7.4	Thr149	11	Activation of caspase-3 and caspase-8[25]
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						His121					His317
						Tyr204
15	Betulin[18]		0.974	0.002	−8.3	Glu123	1	0.869	0.001	−5.5	Thr149	11	Activation of caspase-3[26]
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						His121					His317
						Tyr204
16	Betulinaldehyde[27]		0.976	0.002	−10.3	Glu123	1	0.834	0.001	−7.3	Thr149	11
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						Tyr204					His317
						His121
17	Betulinic acid[18]		0.985	0.002	−7.1	Glu123	1	0.905	0.001	−7.6	Thr149	11	Activation of caspase-3[28]
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						Tyr204					His317
						His121
18	Bryonolic acid[17]		0.736	0.009	−8.6	Glu123	1	0.759	0.002	−7.9	Thr149	11
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						Tyr204					His317
						His121
19	B-sitosterol[29]		0.806	0.005		Glu123	1	0.337	0.099	−6.1	Thr149	11	Activation of caspase-3[30]
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						Tyr204					His317
						His121
20	Canophyllal[18]		0.740	0.009	−10.0	Glu123	1	0.629	0.005	−7.8	Thr149	11
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						Tyr204					His317
						His121
21	Canophyllic acid[18]		0.909	0.003	−9.0	Glu123	1	0.808	0.002	−7.5	Thr149	11
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						Tyr204					His317
						His121
22	CDDO[18]		0.380	0.059	−9.6	Glu123	1	0.562	0.002	−6.3	Thr149	11	Activation of caspase-3 and caspase-8[31]
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						Tyr204					His317
						His121
23	Ceanotdic acid[18]		0.902	0.003	−7.4	Glu123	1	0.825	0.001	−6.7	Thr149	11
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						Tyr204					His317
						His121
24	Celastrol[17]		0.338	0.085	−8.8	Glu123	1	0.526	0.013	−7.4	Thr149	11	Activation of caspase-3 and caspase-8[32]
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						Tyr204					His317
						His121
25	Colubrinic acid[18]		0.944	0.003	−8.0	Glu123	1	0.817	0.002	−6.3	Thr149	11
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						Tyr204					His317
						His121
26	Corosolic acid[18]		0.881	0.004	−8.7	Glu123	1	0.825	0.001	−7.5	Thr149	11	Activation of caspase-3 and caspase-8[33]
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						Tyr204					His317
						His121
27	Coussaric acid[18]		0.875	0.004	−8.4	Glu123	1	0.893	0.001	−6.4	Thr149	11
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						Tyr204					His317
						His121
28	Crotalic acid[18]		0.956	0.003	−7.2	Glu123	1	0.817	0.002	−6.8	Thr149	11
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						Tyr204					His317
						His121
29	Epifriedelanol[18]		0.860	0.004	−8.5	Glu123	1	0.758	0.002	−8.0	Thr149	11
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						Tyr204					His317
						His121
30	Erytdrodiol[18]		0.971	0.002	−8.2	Glu123	1	0.878	0.001	−7.4	Thr149	11
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						Tyr204					His317
						His121
31	Euscaphic acid[18]		0.870	0.004	−8.7	Glu123	1	0.852	0.001	−7.6	Thr149	11
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						Tyr204					His317
						His121
32	Faradiol[18]		0.967	0.002	−5.7	Glu123	1	0.766	0.002	−4.3	Thr149	11
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						Tyr204					His317
						His121
33	Friedelin[18]		0.757	0.008	−9.7	Glu123	1	0.690	0.004	−7.8	Thr149	11	Activation of caspase-3[34]
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						Tyr204					His317
						His121
34	Germanicol[18]		0.881	0.004	−9.1	Glu123	1	0.788	0.002	−7.8	Thr149	11
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						Tyr204					His317
						His121
35	Glycyrrhetinic acid[17]		0.844	0.004	−8.8	Glu123	1	0.794	0.002	−7.8	Thr149	11
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						Tyr204					His317
						His121
36	Glycyrrhizin[18]		0.899	0.003	−9.7	Glu123	1	0.873	0.001	−8.3	Thr149	11
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						Tyr204					His317
						His121
37	Goreishic acid III[18]		0.558	0.020	−8.4	Glu123	1	0.697	0.004	−6.7	Thr149	11
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						Tyr204					His317
						His121
38	Guggulsterone[18]		0.422	0.043	−9.5	Glu123	1	0.534	0.012	−6.6	Thr149	11	Activation of caspase-3 and caspase-8[35]
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						Tyr204					His317
						His121
39	Guluronic acid[36]		0.399	0.051	−4.3	Glu123	1	0.021	0.018	−3.6	Thr149	11
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						Tyr204					His317
						His121
40	Gymnemic acid[8]		0.788	0.006	−7.7	Glu123	1	0.820	0.002	−7.2	Thr149	11	Activation of caspase-3[7]
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						Tyr204					His317
						His121
41	Hederagenin[18]		0.982	0.002	−8.5	Glu123	1	0.918	0.001	−7.0	Thr149	11
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						Tyr204					His317
						His121
42	Hyptatic acid A[37]		0.976	0.002	−9.3	Glu123	1	0.909	0.001	−7.7	Thr149	11
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						Tyr204					His317
						His121
43	Imberbic acid[18]		0.846	0.004	−8.8	Glu123	1	0.804	0.002	−7.3	Thr149	11
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						Tyr204					His317
						His121
44	Lupeol[18]		0.978	0.002	−8.6	Glu123	1	0.865	0.001	−7.8	Thr149	11	Activation of caspase-3[38]
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						Tyr204					His317
						His121
45	Madecassic acid[18]		0.821	0.005	−8.1	Glu123	1	0.778	0.002	−6.0	Thr149	11
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						Tyr204					His317
						His121
46	Madecassoside[18]		0.988	0.001	−9.3	Glu123	1	0.937	0.000	−7.5	Thr149	11
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						Tyr204					His317
						His121
47	Maniladiol[39]		0.987	0.001	−7.6	Glu123	1	0.894	0.001	−7.0	Thr149	11
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						Tyr204					His317
						His121
48	Morolic acid[18]		0.877	0.004	−8.9	Glu123	1	0.806	0.002	−7.2	Thr149	11
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						Tyr204					His317
						His121
49	Moronic acid[18]		0.610	0.016	−8.5	Glu123	1	0.692	0.002	−7.9	Thr149	11
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						Tyr204					His317
						His121
50	Oleanolic acid[17]		0.984	0.002	−8.5	Glu123	1	0.914	0.001	−7.9	Thr149	11
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						Tyr204					His317
						His121
51	Phytolaccagenin[18]		0.964	0.003	−7.0	Glu123	1	0.921	0.001	−6.8	Thr149	11
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						Tyr204					His317
						His121
52	Polygalacic acid[18]		0.980	0.002	−8.3	Glu123	1	0.918	0.001	−6.3	Thr149	11
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						Tyr204					His317
						His121
53	Pristimerin[17]		0.396	0.052	−7.8	Glu123	1	0.639	0.005	−7.3	Thr149	11
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						Tyr204					His317
						His121
54	Rehmannic acid[18]		0.796	0.004	−7.3	Glu123	1	0.743	0.003	−7.0	Thr149	11
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						Tyr204					His317
						His121
55	Rotundic acid[9]		0.886	0.004	−8.6	Glu123	1	0.865	0.001	−7.4	Thr149	11	Activation of caspase-3[40]
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						Tyr204					His317
						His121
56	Taraxasterol[18]		0.729	0.010	−8.8	Glu123	1	0.792	0.002	−6.2	Thr149	11
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						Tyr204					His317
						His121
57	Tingenone[18]		0.279	0.170	−9.7	Glu123	1	0.400	0.052	−7.7	Thr149	11
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						Tyr204					His317
						His121
58	Ursolic acid[18]		0.912	0.003	−8.3	Glu123	1	0.834	0.001	−7.2	Thr149	11	Activation of caspase-3[41]
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						Tyr204					His317
						His121
59	Withaferin A[18]		0.303	0.126	−8.2	Glu123	1	0.299	0.145	−5.9	Thr149	11	Activation of caspase-3[42]
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						Tyr204					His317
						His121
60	Zizyberanalic acid[18]		0.944	0.003	−8.0	Glu123	1	0.817	0.002	−6.9	Thr149	11
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						Tyr204					His317
						His121
61	Caratuberside A[43]		0.857	0.004	−7.9	Glu123	1	0.718	0.003	−7.6	Thr149	11
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						Tyr204					His317
						His121
62	Caratuberside B[43]		0.773	0.007	−7.9	Glu123	1	0.691	0.004	−6.3	Thr149	11
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						Tyr204					His317
						His121
63	Caratuberside C[43]		0.545	0.022	−9.3	Glu123	1	0.445	0.032	−8.0	Thr149	11
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						Tyr204					His317
						His121
64	Caratuberside D[44]		0.495	0.028	−9.4	Glu123	1	0.354	0.084	−7.5	Thr149	11
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						Tyr204					His317
						His121
65	Caratuberside E[44]		0.398	0.052	−9.0	Glu123	1	0.354	0.084	−7.9	Thr149	11
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						Tyr204					His317
						His121
66	Caratuberside F[44]		0.470	0.032	−9.1	Glu123	1	0.435	0.036	−6.8	Thr149	11
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						Tyr204					His317
						His121
67	Caratuberside G[44]		0.689	0.012	−8.5	Glu123	1	0.510	0.016	−7.4	Thr149	11
						Gly122					Pro415
						Rxb300					Tyr412
						Gly165					Ser316
						Tyr204					His317
						His121

CDDO = 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid

Molecular docking with AudoDock Vina

Glycyrrhizin, friedelin, canophyllal, albiziasaponin C, tingenone, withaferin A, and epifriedelanol had highest binding affinity for caspase-3. The binding-free energy for these triterpenoids was − 10.1, −10.0, −10.0, −9.9, −9.8, −9.7, and −9.5 kcal/mol, respectively. While β-boswellic acid, CDDO, canophyllic acid, tingenone, and bryonolic acid showed −10.1, −10.0, −10.0, −9.9, −9.8, −9.7, and −9.5 kcal/mol of binding-free energy, respectively, for caspase-8. Table 1 showed results for docking with all triterpenoids with caspase-3 and caspase-8. We also identified amino acids in the binding pocket on target proteins and type of bonding to map the orientation of ligands in binding pocket [Table 1].

in silico-predicted toxicities were filtered by applying a cutoff of Pa = 0.7 and most significant were reported in Table 2. LD50 for various routes of administration were also predicted and presented.

Table 2

Toxicological profile of triterpenoids

Serial number	Triterpenoids	PASS online toxicological data			Rat acute toxicity LD50 (mg/kg)
		Pa	Pi	Toxicity	IP	IV	Oral	SC
1	3epi Katonic acid	0.971	0.003	Muscle weakness	1917.000	5.478	282.800	297.400
		0.930	0.005	Inflammation
		0.908	0.010	Behavioral disturbance
		0.884	0.005	Nephrotoxic
		0.799	0.026	Hematotoxic
		0.743	0.012	Hypercholesterolemic
2	23 hydroxyursolic acid	0.724	0.016	Hypercholesterolemic	1426.000	7.020	304.800	78.370
3	Albiziasaponin A	0.929	0.005	Inflammation	162.700	20.390	1342.000	116.000
		0.780	0.029	Hematotoxic
		0.754	0.005	Cytotoxic
		0.765	0.018	Nephrotoxic
		0.768	0.028	Behavioral disturbance
		0.745	0.018	Muscle weakness
		0.736	0.019	Embryotoxic
		0.727	0.020	Teratogen
4	Albiziasaponin B	0.950	0.004	Inflammation	134.800	19.660	1211.000	224.000
		0.825	0.022	Hematotoxic
		0.821	0.021	Behavioral disturbance
		0.798	0.014	Muscle weakness
		0.782	0.004	Cytotoxic
		0.788	0.014	Embryotoxic
		0.779	0.014	Teratogen
5	Albiziasaponin C	0.950	0.004	Inflammation	105.700	20.700	430.400	374.600
		0.825	0.022	Hematotoxic
		0.821	0.021	Behavioral disturbance
		0.801	0.013	Nephrotoxic
		0.798	0.014	Muscle weakness
		0.788	0.014	Embryotoxic
		0.782	0.004	Cytotoxic
		0.779	0.014	Teratogen
6	Albiziasaponin D	0.964	0.003	Nephrotoxic	86.090	23.440	1240.000	29.740
		0.946	0.005	Hematotoxic
		0.869	0.002	Cytotoxic
		0.870	0.009	Inflammation
		0.850	0.010	Muscle weakness
		0.841	0.009	Embryotoxic
		0.837	0.009	Teratogen
		0.843	0.018	Behavioral disturbance
7	Albiziasaponin E	0.958	0.003	Nephrotoxic	88.590	22.650	1285.000	27.270
		0.940	0.006	Hematotoxic
		0.852	0.003	Cytotoxic
		0.837	0.011	Muscle weakness
		0.823	0.021	Behavioral disturbance
		0.822	0.011	Embryotoxic
		0.819	0.011	Teratogen
8	Alpha boswellic acid	0.941	0.004	Muscle weakness	1894.000	5.107	374.700	325.800
		0.863	0.009	Inflammation
		0.826	0.009	Nephrotoxic
		0.789	0.026	Behavioral disturbance
		0.743	0.012	Hypercholesterolemic
9	Arjunolic acid	0.855	0.010	Inflammation	1712.000	7.955	1045.000	25.600
		0.778	0.016	Nephrotoxic
		0.727	0.038	Hematotoxic
		0.711	0.038	Behavioral disturbance
10	Asiatic acid				1480.000	8.703	934.700	38.790
11	Asiaticoside	0.864	0.009	Inflammation	132.500	23.930	987.000	80.190
		0.854	0.008	Embryotoxic
		0.818	0.011	Teratogen
		0.752	0.034	Hematotoxic
		0.714	0.037	Behavioral disturbance
12	Barrigenic acid	0.936	0.004	Acidosis. metabolic	369.800	2508.000	2244.000	537.000
		0.927	0.004	Shivering
		0.925	0.004	Red cell aplasia
		0.914	0.002	Skin irritation. corrosive
		0.912	0.008	Euphoria
13	Bartogenic acid	0.751	0.020	Inflammation	1684.000	7.586	978.500	117.800
14	Beta-boswellic acid	0.806	0.013	Muscle weakness	1571.000	5.217	268.200	114.600
		0.728	0.015	Hypercholesterolemic
15	Betulin	0.940	0.005	Inflammation	1253.000	6.229	1935.000	102.700
		0.900	0.003	Irritation
		0.786	0.015	Nephrotoxic
		0.753	0.010	Hypercholesterolemic
16	Betulinaldehyde	0.881	0.003	Irritation	1803.000	4.011	2086.000	263.700
		0.803	0.015	Inflammation
17	Betulinic acid	0.933	0.005	Inflammation	1742.000	5.972	2253.000	248.500
		0929	0.003	Irritation
		0.871	0.015	Behavioral disturbance
		0.829	0.009	Nephrotoxic
		0.762	0.008	Hypercholesterolemic
18	Bryonolic acid	0.952	0.004	Muscle weakness	2301.000	6.978	426.400	1032.000
		0.881	0.008	Inflammation
		0.867	0.006	Nephrotoxic
		0.850	0.017	Behavioral disturbance
		0.828	0.004	Hypercholesterolemic
		0.770	0.031	Hematotoxic
19	B−sitosterol	0.931	0.008	Conjunctivitis	1765.000	6.052	788.500	602.500
		0.926	0.008	Sleep disturbance
		0.907	0.004	Teratogen
		0.903	0.005	Embryotoxic
		0.906	0.008	Ocular toxicity
20	Canophyllal				1594.000	11.770	2698.000	2698.000
21	Canophyllic acid	0.810	0.004	Hypercholesterolemic	1981.000	27.870	4711.000	1328.000
		0.817	0.013	Inflammation
		0.778	0.016	Nephrotoxic
		0.766	0.029	Behavioral disturbance
		0.724	0.007	Irritation
22	CDDO				756.300	6.880	1478.000	181.700
23	Ceanothic acid	0.838	0.011	Inflammation	1473.000	7.363	1795.000	78.960
		0.789	0.005	Irritation
		0.716	0.026	Nephrotoxic
24	Celastrol	0.812	0.011	Nephrotoxic	1725.000	9.682	317.100	742.800
		0.807	0.013	Muscle weakness
		0.710	0.024	Inflammation
25	Colubrinic acid	0.807	0.014	Inflammation	1413.000	5.347	1342.000	127.300
		0.750	0.006	Irritation
26	Corosolic acid	0.787	0.016	Inflammation	1428.000	6.478	284.600	98.930
27	Coussaric acid	0.800	0.013	Nephrotoxic	1336.000	7.511	198.800	47.590
		0.723	0.020	Teratogen
		0.729	0.035	Behavioral disturbance
28	Crotalic acid	0.912	0.005	Muscle weakness	1662.000	5.205	252.700	673.900
		0.890	0.012	Behavioral disturbance
		0.860	0.009	Inflammation
		0.849	0.004	Irritation
		0.847	0.003	Hypercholesterolemic
		0.821	0.010	Nephrotoxic
		0.771	0.031	Hematotoxic
29	Epifriedelanol	0.801	0.004	Hypercholesterolemic	2016.000	28.520	4438.000	1268.000
		0.770	0.005	Irritation
		0.765	0.018	Inflammation
		0.723	0.020	Muscle weakness
		0.715	0.026	Nephrotoxic
30	Erythrodiol	0.926	0.006	Inflammation	1495.000	5.072	590.400	94.840
		0.886	0.007	Muscle weakness
		0.860	0.011	Weakness
		0.803	0.013	Nephrotoxic
		0.733	0.014	Hypercholesterolemic
31	Euscaphic acid				1060.000	7.899	916.600	101.700
32	Faradiol	0.968	0.002	Irritation	1461.000	6.108	1843.000	517.200
		0.865	0.009	Inflammation
		0.843	0.010	Muscle weakness
		0.794	0.013	Teratogen
		0.771	0.015	Embryotoxic
		0.732	0.034	Behavioral disturbance
		0.708	0.008	Sensitization
33	Friedelin	0.756	0.009	Hypercholesterolemic	1736.000	33.690	4245.000	3218.000
		0.760	0.019	Inflammation
		0.759	0.019	Nephrotoxic
		0.711	0.038	Behavioral disturbance
		0.702	0.011	Cataract
34	Germanicol	0.820	0.012	Muscle weakness	1806.000	15.770	1854.000	1240.000
		0.783	0.006	Hypercholesterolemic
35	Glycyrrhetinic acid	0.988	0.002	Weakness	2133.000	5.810	269.600	239.300
		0.985	0.002	Muscle weakness
		0.946	0.005	Inflammation
		0.940	0.004	Nephrotoxic
		0.932	0.007	Behavioral disturbance
		0.882	0.014	Hematotoxic
		0.872	0.009	Hypotension
		0.852	0.003	Cardiodepressant
36	Glycyrrhizin	0.996	0.001	Muscle weakness	192.200	23.220	2097.000	144.200
		0.995	0.002	Inflammation
		0.990	0.002	Nephrotoxic
		0.983	0.003	Behavioral disturbance
		0.965	0.004	Hematotoxic
		0.948	0.005	Drowsiness
		0.895	0.012	Sleep disturbance
		0.849	0.011	Hypertensive
		0.776	0.007	Hypercholesterolemic
37	Goreishic acid III	0.723	0.008	Sensitization	1132.000	6.849	283.500	90.770
38	Guggulsterone	0.856	0.008	Teratogen	875.900	33.210	2550.000	3396.000
		0.845	0.009	Embryotoxic
		0.844	0.014	Necrosis
		0.832	0.017	Hepatotoxic
		0.819	0.013	Excitability
		0.803	0.018	Reproductive dysfunction
		0.791	0.007	Optic neuropathy
		0.796	0.008	Weight gain
		0.791	0.025	Behavioral disturbance
		0.781	0.019	Neurotoxic
39	Guluronic acid	0.929	0.006	Reproductive dysfunction	119.900	436.400	1.009	761.200
		0.814	0.023	Diarrhea
		0.813	0.022	Behavioral disturbance
		0.790	0.025	Pure red cell aplasia
		0.745	0.021	Hematemesis
		0.740	0.019	Hyperglycemic
		0.734	0.012	Endocrine disruptor
		0.731	0.020	Occult bleeding
40	Gymnemic acid	0.969	0.003	Muscle weakness	217.200	20.090	1775.000	34.460
		0.955	0.003	Teratogen
		0.948	0.003	Embryotoxic
		0.909	0.007	Inflammation
		0.828	0.021	Hematotoxic
		0.809	0.004	Cytotoxic
		0.740	0.022	Nephrotoxic
		0.732	0.034	Behavioral disturbance
41	Hederagenin	0.855	0.010	Inflammation	1631.000	6.869	316.700	58.190
		0.800	0.013	Nephrotoxic
		0.789	0.015	Muscle weakness
		0.746	0.032	Behavioral disturbance
		0.739	0.013	Hypercholesterolemic
		0.738	0.023	Hypotension
42	Hyptatic acid A	0.817	0.013	Inflammation	1330.000	9.343	850.800	177.200
		0.708	0.027	Nephrotoxic
		0.703	0.039	Behavioral disturbance
43	Imberbic acid	0.968	0.003	Muscle weakness	1964.000	5.893	242.300	63.530
		0.929	0.005	Inflammation
		0.883	0.013	Behavioral disturbance
		0.864	0.006	Nephrotoxic
		0.783	0.029	Hematotoxic
44	Lupeol	0.948	0.003	Irritation	1684.000	5.867	2888.000	786.900
		0.901	0.007	Inflammation
		0.821	0.021	Behavioral disturbance
		0.794	0.014	Nephrotoxic
		0.751	0.010	Hypercholesterolemic
45	Madecassic acid				1191.000	8.980	1069.000	52.300
46	Madecassoside	0.858	0.008	Embryotoxic	127.300	24.410	733.800	479.200
		0.842	0.011	Inflammation
		0.822	0.011	Teratogen
		0.767	0.028	Behavioral disturbance
		0.725	0.038	Hematotoxic
47	Maniladiol	0.947	0.004	Muscle weakness	1680.000	3.987	1163.000	318.900
		0.917	0.006	Inflammation
		0.866	0.015	Behavioral disturbance
		0.821	0.010	Nephrotoxic
48	Morolic acid	0.875	0.008	Muscle weakness	1647.000	8.676	1984.000	1087.000
		0.793	0.005	Hypercholesterolemic
		0.796	0.014	Nephrotoxic
		0.760	0.019	Inflammation
		0.735	0.034	Behavioral disturbance
49	Moronic acid	0.728	0.015	Hypercholesterolemic	1291.000	7.758	2107.000	1399.000
50	Oleanolic acid	0.917	0.006	Inflammation	1749.000	5.124	369.600	146.500
		0.886	0.007	Muscle weakness
		0.868	0.015	Behavioral disturbance
		0.842	0.008	Nephrotoxic
		0.743	0.012	Hypercholesterolemic
		0.734	0.037	Hematotoxic
51	Phytolaccagenin	0.836	0.011	Inflammation	1531.000	10.210	920.600	264.200
		0.765	0.018	Nephrotoxic
		0.739	0.019	Muscle weakness
		0.732	0.034	Behavioral disturbance
52	Polygalacic acid	0.825	0.013	Inflammation	1629.000	7.522	828.300	38.460
		0.727	0.024	Nephrotoxic
53	Pristimerin	0.710	0.021	Muscle weakness	1400.000	7.212	1084.000	1048.000
		0.703	0.028	Nephrotoxic
54	Rehmannic acid	0.956	0.004	Hepatotoxic	1169.000	5.029	209.600	74.740
55	Rotundic acid	0.817	0.013	Inflammation	1249.000	7.539	229.000	78.980
		0.743	0.022	Nephrotoxic
		0.740	0.033	Behavioral disturbance
56	Taraxasterol	0.937	0.003	Irritation	1584.000	4.612	1619.000	877.100
		0.737	0.023	Nephrotoxic
		0.713	0.019	Hypercholesterolemic
57	Tingenone	0.730	0.024	Nephrotoxic	1488.000	7.469	2268.000	1164.000
58	Ursolic acid	0.788	0.016	Inflammation	1459.000	5.376	265.000	146.500
		0.728	0.015	Hypercholesterolemic
		0.719	0.025	Nephrotoxic
59	Withaferin A				807.200	13.930	1310.000	267.700
60	Zizyberanalic acid	0.807	0.014	Inflammation	1413.000	5.347	1342.000	127.300
		0.750	0.006	Irritation
61	Caratuberside A	0.982	0.002	Neurotoxic	68.810	21.640	80.290	69.880
		0.978	0.003	Dyspnea
		0.955	0.003	Teratogen
		0.952	0.003	Embryotoxic
		0.949	0.005	Behavioral disturbance
		0.948	0.004	Emetic
		0.945	0.004	Excitability
		0.934	0.003	Necrosis
		0.918	0.007	Reproductive dysfunction
62	Caratuberside B	0.974	0.002	Neurotoxic	64.310	24.150	69.160	43.140
		0.958	0.004	Nausea
		0.949	0.005	Hematotoxic
		0.938	0.005	Emetic
		0.920	0.004	Necrosis
		0.921	0.009	Behavioral disturbance
		0.904	0.004	Teratogen
		0.900	0.005	Embryotoxic
63	Caratuberside C	0.967	0.004	Hematotoxic	31.050	13.840	76.930	29.410
		0.965	0.003	Neurotoxic
		0.950	0.003	Teratogen
		0.947	0.003	Embryotoxic
		0.947	0.005	Behavioral disturbance
		0.940	0.005	Nausea
		0.896	0.007	Necrosis
		0.869	0.008	Dyspnea
64	Caratuberside D	0.952	0.003	Neurotoxic	95.900	15.200	78.220	28.230
		0.952	0.005	Hematotoxic
		0.947	0.005	Behavioral disturbance
		0.937	0.005	Nausea
		0.907	0.005	Embryotoxic
		0.902	0.005	Teratogen
		0.897	0.007	Necrosis
		0.872	0.008	Dyspnea
65	Caratuberside E	0.955	0.005	Hematotoxic	20.950	14.680	86.890	29.950
		0.946	0.003	Neurotoxic
		0.919	0.007	Nausea
		0.912	0.010	Behavioral disturbance
		0.871	0.008	Dyspnea
		0.870	0.010	Necrosis
		0.864	0.012	Emetic
		0.849	0.008	Teratogen
66	Caratuberside F	0.974	0.004	Hematotoxic	54.050	17.210	86.540	22.040
		0.963	0.003	Neurotoxic
		0.949	0.005	Nausea
		0.935	0.006	Behavioral disturbance
		0.908	0.008	Emetic
		0.903	0.005	Dyspnea
		0.896	0.005	Teratogen
		0.892	0.005	Embryotoxic
67	Caratuberside G	0.981	0.002	Neurotoxic	83.040	14.760	255.000	25.450
		0.966	0.002	Teratogen
		0.966	0.002	Embryotoxic
		0.962	0.004	Behavioral disturbance
		0.959	0.004	Hematotoxic
		0.958	0.004	Nausea
		0.890	0.008	Necrosis
		0.884	0.009	Emetic

IP = Intraperitoneal, IV = Intravenous, SC = Subcutaneous, CDDO = 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid

Discussion

Triterpenoids are metabolites of isopentenyl pyrophosphate oligomers and are ubiquitously distributed throughout the plant kingdom.[45] These are a class of chemical compounds composed of three terpene units with the molecular formula C30H48.[46] Naturally-occurring[47], as well as synthetic triterpenoids[4849], have wide spectrum of biological and pharmacological effects, including anti-inflammatory, antibacterial, antiviral, hepatoprotective, gastroprotective, anti-ulcer, cardiovascular, hypolipidemic, antiatherosclerotic, immunoregulatory, anticancer, and cancer preventive activities.[50]

Recently, adulteration, content variation, and hence safety of herbal medicaments or formulations forced researchers to search for alternative strategy to isolate, standardize, and formulate herbal drugs.[51] Ancient approach used mixture of multiple herbal components as a drug,[52] but modern medical science shifted the approach to isolate single component and establish the pharmacological significance of it.[53] In our study, we pushed this approach further and carried out in silico testing of these herbal components, i.e., triterpenoids to predict the pharmacological activities and toxicities of these compounds.

Through literature search, we identified various natural and synthetic triterpenoids and obtained their structures [Table 1]. These triterpenoids were subjected for pharmacological activity prediction using PASS Online software and those showing Pa >0.7 either for anticancer activity, apoptogenic potential, or caspase-3 and/or caspase-8 stimulation were selected for further processing. We obtained 67 triterpenoids, the activity prediction data of which is represented in Table 1. Among these 67 compounds, 15 were previously reported for caspase-3 and/or caspase-8 stimulation activity with Pa of above 0.7 (11 out of 15 for caspase-3 and 9 out of 15 for caspase-8). This ascertains the vitality of in silico predictions to determine lead compounds from the herbal origin.

Further, we processed these compounds for docking against caspase-3 and caspase-8 through AutoDock Vina. We identified the binding pocket on proteins and docking was done targeting these pockets. The minimum binding-free energy of −10.0, −9.9, −9.8, and −9.5 were shown by friedelin, tingenone, albiziasaponin A, and albiziasaponin C, respectively, for caspases-3, and -11, -9.6, -9.6, -9.4 by β-boswellic acid, bryonolic acid, canophyllic acid, and CDDO, respectively, for caspase-8 [Table 1]. Most of these compounds are reported for their anticancer activity, but the exact mechanism is still remained to be established. The results obtained in our study pointed toward the apoptogenic potential through stimulation of either caspase-3 or caspase-8 as a mechanism for these compounds, and future studies may target these proteins.

Safety of triterpenoid saponins always remained as a concern for authorities,[5455] especially in Western countries. Uses of these saponins are even prohibited in some countries, particularly in Central and Southern America,[55] hence we addressed this issue by predicting the toxicities of triterpenoids selected for our study through GUSAR method. The results obtained are reported in Table 2. We even predicted LD50 of these triterpenoids in rats by various routes of administration. Most of these triterpenoids were predicted to be inflammatory in nature and produces hematotoxicity. Various previous studies reported triterpenoid saponins as hematotoxic[545657] which supports results we obtained in our study through in silico predictions.

Conclusion

The predictions of caspase-stimulant activity (along with docking with caspase-3 and 8) and toxicity profile of triterpenoids helped in lead compound identification, e.g., albiziasaponin A, albiziasaponin C, caratuberoside A, and canophyllal for further in vitro and in vivo studies. Results obtained in our study will be helpful to researchers in planning further studies with these triterpenoids.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.