The antagonist binds deeper into the CB2R pocket than the partial agonist
Crystal structure of antagonist AM10257-bound CB2R (PDB ID: 5ZTY) [13] was used as a template to build the homology model of CB2R. During the mode building, intracellular loop 3 (IL3) was inserted and the cocrystal ligand, antagonist AM10257 was retained. The model comprises seven transmembranes (7TM) helices connected by loops and the C-terminal region only. The antagonist AM10257 is bound to CB2R with its substituted core pyrazole group residing among TM2, TM3, and TM7 and forms mainly hydrophobic interactions with residues at ECL2, TM2, TM3, TM4, and TM6 (Fig. 3A). The docking pose of the partial agonist Δ9-THC reveals that the tetrahydro-6H-benzo[c]chromene ring system is located within the main hydrophobic pocket, with an alkyl chain at C3 of the resorcinol moiety buried deep inside the tunnel (Fig. 3B) like described elsewhere [35]. This shorter pentyl chain of Δ9-THC only partially occupies the hydrophobic tunnel compared with the same chain in the antagonist AM10257 having an extra hydroxyl group (Fig. 3C).
The Δ9-thc Induces Minor Conformational Changes At Cb2r
The conformational changes of the antagonist AM10257 bound CB2R and partial agonist Δ9-THC bound CB2R are compared in terms of root-mean-square deviation (RMSD) profile of the specific regions of the receptor. The antagonist system maintains the initial conformation of the TMD. This is manifested in the RMSD of the TM6 which appears to be the same upon alignment based on the TMD with TM6 (Fig. 4A) and without the TM6 (Fig. 4B). On the other hand, RMSD of the TM6 increases in the partial agonist system upon alignment based on TM1-5& TM7 (Figs. 4C and D), suggesting movement of the TM6. This movement of TM6 was also analyzed in the rest of the two trajectories for each system. All the 3 trajectories for the antagonist system maintain the conformation of the TMD (Figure S1). However, only two out of the 3 trajectories of the partial agonist system show the outward movement of TM6 from the 7TM bundle (Figure S2).
The helical content of both antagonist and partial agonist systems are conserved through the simulation (Figure S3), and no significant difference in the root-mean-square fluctuation (RMSF) profiles of the TMD between the two systems. However, intracellular loop 3 (IL3)—the missing loop in the CB2R structure (5ZTY) inserted during model building, shows high fluctuation (Figure S4).
The Ligands Form Different Interactions With Cb2r
The AM10257, acting as a competitive antagonist of CB2R spans the CB2R pocket with its core pyrazole ring and a three-arm scaffold assuming a constrained binding pose. The antagonist forms mainly hydrophobic interactions with CB2R and an H-bond between the hydroxyl group of the alkyl chain and T114 at CB2R persisting in more than 60% of the simulation (Fig. 5A). On the other hand, despite binding to the same site as the antagonist AM10257, the partial agonist Δ9-THC, via hydroxyl substituent on its polycyclic ring, forms 2 H-bonds with K109 and S90 in about 35% of the simulation. Other interactions formed are mainly hydrophobic, however, the alkyl chain does not appear to be involved in persistent interactions with the receptor (Fig. 5B). The other two trajectories for the respective systems show similar interaction patterns (Figure S5 and 6). Also, the two ligands show different torsion angle distribution, with the antagonist AM10257 displaying high flexibility, especially at the alkyl linker region (Figure S7). This allows the linker to be buried completely in the hydrophobic pocket of CB2R.
The toggle switch” W6.48(258) experiences an upward shift in the partial agonist system
Trajectory clustering analysis was performed to identify the dominant structure through the simulation. The most abundant clusters are found to be 62% and 64% for the antagonist and partial agonist systems, respectively. Their representative structures were superimposed with both crystal structures of the antagonist AM10257 bound CB2R (5ZTY) and agonist bound CB2R (6KPC) to look for conformational changes, particularly, at TM6 and the “toggle switch” W6.48(258). Compared to the crystal structure of the antagonist bound CB2R, the antagonist system does not show significant changes at both the TM6 and W6.48(258) (Fig. 6A). However, when compared to the agonist bound crystal structure, the TM6 does not show significant change, and the W6.48(258) does not shift upward in the antagonist system (Fig. 6B). On the other hand, the TM6 slightly moves out of the 7TM in the partial agonist system and W6.48(258) experiences a strong upward shift like in the crystal structure of the agonist bound CB2R (Figs. 6C-F). While the W6.48(258) dihedral angle is maintained (between 18 and 27 degrees) through the simulation in the antagonist system (Fig. 7A), it deviates high (between − 15 and 40 degrees) between 530 and 720 ns (Fig. 7B). Even though this conformational change of the W6.48(258) appears to be transient, its magnitude is reflected in the representative structure of the most abundant cluster of the partial agonist bound CB2R. Consistent trends can be seen in all the 3 trajectories of the antagonist system and at least a trajectory of the partial agonist system (Figures S8-10).
Dynamics Network Model Identifies Possible Signal Transduction Path
Dynamical network models were generated to understand the relationship between different structural units (referred to as communities) of CB2R following ligand binding and to identify possible signal transduction paths. For the antagonist system, the weighted network does show high correlations between structural units around TM6 (Fig. 8A) that are grouped into 11 communities (Fig. 8B). Due to the binding of the antagonist deeper in the CB2R pocket, a possible signal transduction path comprises only residues T114 and T116 at TM3 relaying the signal to the “toggle switch” W6.48(258) at the TM6 (Fig. 8C). In the case of the partial agonist system, thicker connections are observed at the extracellular site of TM5 and TM6 (Fig. 8D), suggesting correlated movement of these regions. A total of 12 communities are identified (Fig. 8E), and a path comprising S90 and Y87 at TM2; L289 and C288 at TM7, relaying the signal to W6.48(258) at the TM6. Also, while the communication paths differ among the 3 trajectories of the antagonist system (Figure S11), the same path is identified in all the 3 trajectories of the partial agonist (Figure S12). The dynamic network model reveals different community arrangements between the two systems, the possible correlation between the movement of TM5 and TM6 in the partial agonist system, and a longer communication between the partial agonist Δ9-THC and the “toggle switch” W6.48(258).
Structure-based Virtual Screening, Md Simulation, And Mmgbsa Evaluation
The three most abundant conformations derived from MD simulation of partial agonist Δ9-THC bound CB2R were used for virtual screening of ZINC15 “Druglike” library. The top 30 hits (10 hits against each confirmation), each complexed with the CB2R were submitted to 200 ns MD simulation. A total of 17 diverse compounds (Fig. 9) were found to have a higher MM-GBSA binding energy score than the partial agonist Δ9-THC (Table 1) and are therefore considered as the initial hit partial agonists of CB2R. Their binding poses are compared to that of the agonist AM12033 in the active crystal structure 6KPC. Even though they all bind to the same agonist binding site, they show slightly different modes compared to the agonist AM12033 (Figure S13).
Table 1
Ligand binding energy of the top 30 hits compared to the reference compound Δ9-THC calculated using docking and MD simulation properties. The virtual screening was conducted against 3 conformations: conformation 1 (blue); conformation 2 (orange); conformation 3 (green).
#
|
Compound
|
Docking Score (kcal/mol)
|
VDW (kcal/mol)
|
ELE (kcal/mol)
|
Hydrophobic (kcal/mol)
|
MM-GBSA
(kcal/mol)
|
Receptor RMSD1 (Å)
|
Ligand RMSD1 (Å)
|
Ref.
|
Δ9-THC
|
-11.7
|
-48.2 ± 2.6
|
-6.0 ± 3.0
|
-61.4 ± 3.8
|
-115.6 ± 6.7
|
|
|
1
|
ZINC000009762581
|
-16.7
|
-71.8 ± 4.4
|
-14.0 ± 2.9
|
-58.3 ± 5.0
|
-144.1 ± 9.6
|
2.8 ± 0.2
|
0.9 ± 0.1
|
2
|
ZINC000096124070
|
-16.4
|
-66.6 ± 5.9
|
-15.7 ± 4.6
|
-40.8 ± 2.7
|
-123.0 ± 8.3
|
2.8 ± 0.6
|
1.2 ± 0.3
|
3
|
ZINC000534577320
|
-16.1
|
-59.3 ± 2.9
|
-13.2 ± 3.4
|
-47.6 ± 2.6
|
-120.1 ± 8.8
|
4.3 ± 0.6
|
0.7 ± 0.2
|
4
|
ZINC000017192627
|
-16.1
|
-68.4 ± 4.8
|
-30.5 ± 4.1
|
-43.7 ± 3.1
|
-142.6 ± 7.9
|
3.9 ± 0.9
|
1.8 ± 0.2
|
5
|
ZINC000096482611
|
-15.7
|
-49.8 ± 3.6
|
-4.8 ± 3.9
|
-38.2 ± 3.2
|
-92.8 ± 6.9
|
2.5 ± 0.5
|
1.1 ± 0.3
|
6
|
ZINC000002977189
|
-15.6
|
-54.6 ± 2.6
|
-6.7 ± 3.0
|
-45.2 ± 2.8
|
-106.5 ± 4.5
|
2.6 ± 0.5
|
1.4 ± 0.2
|
7
|
ZINC000005895757
|
-15.5
|
-52.4 ± 3.9
|
-22.9 ± 3.2
|
-22.4 ± 1.9
|
-97.7 ± 4.8
|
2.7 ± 0.5
|
0.7 ± 0.1
|
8
|
ZINC000010130639
|
-15.5
|
-49.8 ± 3.5
|
-9.1 ± 6.7
|
-68.5 ± 5.2
|
-127.4 ± 10.5
|
2.7 ± 0.5
|
0.7 ± 0.2
|
9
|
ZINC000005890991
|
-15.3
|
-53.4 ± 3.3
|
-19.5 ± 4.4
|
-26.8 ± 1.8
|
-99.8 ± 6.1
|
3.1 ± 0.6
|
0.6 ± 0.1
|
10
|
ZINC000409265923
|
-15.1
|
-67.4 ± 2.7
|
-13.0 ± 3.1
|
-46.1 ± 2.8
|
-126.5 ± 5.2
|
3.1 ± 0.7
|
1.6 ± 0.2
|
11
|
ZINC000067457850
|
-15.0
|
-52.8 ± 2.8
|
-7.9 ± 2.7
|
-41.2 ± 2.6
|
-102.0 ± 6.3
|
2.1 ± 0.2
|
1.0 ± 0.2
|
12
|
ZINC000082087170
|
-14.6
|
-52.4 ± 2.9
|
-21.1 ± 9.1
|
-35.1 ± 1.7
|
-108.7 ± 10.6
|
3.0 ± 0.6
|
1.1 ± 0.2
|
13
|
ZINC000004403342
|
-14.4
|
-86.3 ± 4.2
|
-23.3 ± 8.4
|
-84.5 ± 6.3
|
-194.1 ± 14.9
|
2.8 ± 0.4
|
1.5 ± 0.3
|
14
|
ZINC000089836703
|
-14.4
|
-51.7 ± 2.5
|
-9.7 ± 2.9
|
-39.6 ± 1.8
|
-101.0 ± 4.6
|
2.4 ± 0.3
|
2.2 ± 0.2
|
15
|
ZINC000013548332
|
-14.3
|
-62.4 ± 3.5
|
-12.7 ± 3.4
|
-47.9 ± 2.8
|
-123.0 ± 6.6
|
2.3 ± 0.3
|
2.2 ± 0.5
|
16
|
ZINC000006752723
|
-14.3
|
-59.6 ± 3.2
|
-21.7 ± 4.1
|
-47.3 ± 4.1
|
-128.6 ± 8.1
|
2.4 ± 04
|
0.7 ± 0.2
|
17
|
ZINC000218260651
|
-14.3
|
-57.4 ± 4.5
|
-15.3 ± 4.6
|
-44.1 ± 3.4
|
-116.8 ± 10.2
|
2.9 ± 0.4
|
0.3 ± 0.1
|
18
|
ZINC000095527098
|
-14.3
|
-58.4 ± 3.8
|
-9.2 ± 6.3
|
-46.7 ± 4.3
|
-114.4 ± 11.8
|
3.0 ± 0.4
|
1.3 ± 0.4
|
19
|
ZINC000008791953
|
-14.3
|
-60.6 ± 4.9
|
-9.2 ± 4.2
|
-43.1 + 3.6
|
-112.9 ± 8.6
|
2.6 ± 0.5
|
0.9 ± 0.1
|
20
|
ZINC000257304951
|
-14.2
|
-59.8 ± 3.9
|
-12.7 ± 5.0
|
-43.9 ± 2.7
|
-116.3 ± 7.5
|
2.5 ± 0.4
|
1.9 ± 0.4
|
21
|
ZINC000013475970
|
-16.5
|
-113.1 ± 5.0
|
-29.5 ± 6.3
|
-75.4 ± 4.8
|
-218.0 ± 12.4
|
4.3 ± 0.9
|
0.9 ± 0.2
|
22
|
ZINC000002765499
|
-15.8
|
-48.6 ± 2.5
|
-10.7 ± 2.8
|
-37.5 ± 2.3
|
-96.9 ± 5.2
|
3.0 ± 0.6
|
1.1 ± 0.2
|
23
|
ZINC000408739159
|
-15.7
|
-66.1 ± 5.0
|
-11.8 ± 10.1
|
-48.9 ± 3.3
|
-126.8 ± 12.0
|
2.8 ± 0.6
|
1.6 ± 0.2
|
24
|
ZINC000013356709
|
-15.6
|
-65.5 ± 3.7
|
-13.7 ± 6.3
|
-54.9 ± 2.9
|
-134.9 ± 9.0
|
2.9 ± 0.4
|
1.9 ± 0.4
|
25
|
ZINC000252533065
|
-15.3
|
-59.4 ± 3.2
|
-13.7 ± 3.1
|
-41.9 ± 2.3
|
-115.0 ± 6.2
|
3.0 ± 0.6
|
1.2 ± 0.3
|
26
|
ZINC000089917273
|
-15.3
|
-52.4 ± 3.3
|
-16.6 ± 5.3
|
-47.0 ± 5.2
|
-116.0 ± 1.1
|
3.5 ± 0.7
|
1.6 ± 0.5
|
27
|
ZINC000001464338
|
-15.2
|
-54.5 ± 3.7
|
-16.7 ± 4.4
|
-40.6 ± 2.9
|
-111.8 ± 8.7
|
3.0 ± 0.6
|
0.6 ± 0.1
|
28
|
ZINC000002687799
|
-15.1
|
-65.6 ± 3.1
|
-18.5 ± 4.2
|
-49.4 ± 3.4
|
-133.5 ± 8.5
|
2.7 ± 0.3
|
0.7 ± 0.2
|
29
|
ZINC000012628414
|
-15.0
|
-59.3 ± 4.4
|
-14.2 ± 6.0
|
-42.6 ± 3.8
|
-116.1 ± 8.0
|
2.7 ± 0.4
|
2.4 ± 0.7
|
30
|
ZINC000072477563
|
-15.0
|
-60.7 ± 3.1
|
-14.5 ± 4.1
|
-39.1 ± 3.2
|
-114.3 ± 8.5
|
3.1 ± 0.9
|
2.4 ± 0.4
|
1. Based on the snapshots from the last 10 ns simulation. Based on MM-GBSA scores < 115.6 Kcal/mol and the top compounds are represented in bold font.
The Final Hits Show Good Admet Properties
The final hits were selected based on ADMET properties predicted using the SwissADME server. A total of 9 compounds selected as potential partial agonists of CB2R were predicted to demonstrate high gastrointestinal (GI) absorption, non-blood-brain barrier penetration, less chance of inhibiting liver enzymes associated with drug metabolism, 0 violation of Lipinski’s “rule of five” and 0 PAINS alert (Table 2). The selected compounds mustn’t pass the blood-brain blood, such that they may target only CB2R in the immune cells but not CB1R which is predominantly expressed in the brain. The ADMET properties of the individual hits are available in Figure S14.
Table 2
Druglike and ADMET properties of the top 30 compounds predicted using SwissADME server. Compounds shown in bold have higher MM-GBSA energy scores than that of Δ9-THC. Compounds highlighted in green have good ADMET properties but do not pass the blood-brain barrier (BBB), such that they target only CB2R in the immune cells but not CB1R which is predominantly expressed in the brain.
S/N
|
Compound
|
GI absorption
|
BBB permeant
|
CYP1A2 inhibitor
|
CYP2C19 inhibitor
|
CYP2C9 inhibitor
|
CYP2D6 inhibitor
|
CYP3A4 inhibitor
|
Lipinksi rule
|
PAINS
|
Ref.
|
Δ9-THC
|
High
|
Yes
|
No
|
Yes
|
Yes
|
Yes
|
Yes
|
Yes; 1 violation: MLOGP > 4.15
|
0 alert
|
Ref.
|
CBD
|
High
|
Yes
|
No
|
Yes
|
Yes
|
Yes
|
Yes
|
Yes; 1 violation: MLOGP > 4.15
|
0 alert
|
Ref.
|
CB2R-selective agonist AM1710
|
High
|
No
|
No
|
Yes
|
Yes
|
Yes
|
No
|
Yes, 0 violation
|
0 alert
|
Ref.
|
CB2R-selective agonist JWH-133
|
Low
|
No
|
No
|
Yes
|
Yes
|
No
|
No
|
Yes; 1 violation: MLOGP > 4.15
|
0 alert
|
1
|
ZINC000009762581
|
High
|
No
|
Yes
|
Yes
|
Yes
|
Yes
|
No
|
Yes, 0 violation
|
0 alert
|
2
|
ZINC000096124070
|
High
|
No
|
Yes
|
No
|
No
|
No
|
No
|
Yes, 0 violation
|
0 alert
|
3
|
ZINC000534577320
|
High
|
Yes
|
Yes
|
No
|
Yes
|
Yes
|
Yes
|
Yes, 0 violation
|
0 alert
|
4
|
ZINC000017192627
|
Low
|
No
|
No
|
Yes
|
No
|
No
|
No
|
Yes, 0 violation
|
0 alert
|
5
|
ZINC000096482611
|
High
|
No
|
No
|
Yes
|
Yes
|
Yes
|
Yes
|
Yes, 0 violation
|
0 alert
|
6
|
ZINC000002977189
|
High
|
No
|
Yes
|
Yes
|
Yes
|
No
|
Yes
|
Yes, 0 violation
|
1 alert: ene_six_hetA
|
7
|
ZINC000005895757
|
High
|
No
|
No
|
No
|
No
|
No
|
No
|
Yes, 0 violation
|
0 alert
|
8
|
ZINC000010130639
|
High
|
No
|
Yes
|
Yes
|
Yes
|
No
|
Yes
|
Yes, 0 violation
|
1 alert: ene_six_hetA
|
9
|
ZINC000005890991
|
High
|
No
|
No
|
No
|
No
|
No
|
Yes
|
Yes, 0 violation
|
0 alert
|
10
|
ZINC000409265923
|
High
|
No
|
No
|
No
|
Yes
|
No
|
Yes
|
Yes, 0 violation
|
1 alert: ene_six_hetA
|
11
|
ZINC000067457850
|
High
|
Yes
|
Yes
|
Yes
|
Yes
|
Yes
|
Yes
|
Yes, 0 violation
|
0 alert
|
12
|
ZINC000082087170
|
High
|
No
|
No
|
No
|
No
|
No
|
No
|
Yes, 0 violation
|
0 alert
|
13
|
ZINC000004403342
|
Low
|
No
|
Yes
|
No
|
No
|
Yes
|
Yes
|
Yes, 0 violation
|
0 alert
|
14
|
ZINC000089836703
|
High
|
No
|
No
|
No
|
No
|
Yes
|
No
|
Yes, 0 violation
|
0 alert
|
15
|
ZINC000013548332
|
High
|
No
|
No
|
No
|
No
|
No
|
Yes
|
Yes, 0 violation
|
1 alert: anil_di_alk_E
|
16
|
ZINC000006752723
|
High
|
No
|
No
|
No
|
No
|
No
|
Yes
|
Yes, 0 violation
|
1 alert: anil_di_alk_E
|
17
|
ZINC000218260651
|
High
|
Yes
|
Yes
|
Yes
|
No
|
No
|
No
|
Yes, 0 violation
|
1 alert: catechol_A
|
18
|
ZINC000095527098
|
High
|
No
|
Yes
|
Yes
|
No
|
No
|
No
|
Yes, 0 violation
|
1 alert: catechol_A
|
19
|
ZINC000008791953
|
High
|
No
|
No
|
Yes
|
Yes
|
Yes
|
Yes
|
Yes, 0 violation
|
0 alert
|
20
|
ZINC000257304951
|
High
|
No
|
No
|
Yes
|
Yes
|
Yes
|
Yes
|
Yes, 0 violation
|
0 alert
|
21
|
ZINC000013475970
|
High
|
No
|
No
|
No
|
No
|
No
|
Yes
|
Yes, 0 violation
|
0 alert
|
22
|
ZINC000002765499
|
High
|
Yes
|
No
|
Yes
|
Yes
|
Yes
|
Yes
|
Yes, 0 violation
|
0 alert
|
23
|
ZINC000408739159
|
High
|
No
|
No
|
Yes
|
Yes
|
Yes
|
Yes
|
Yes, 0 violation
|
0 alert
|
24
|
ZINC000013356709
|
High
|
No
|
No
|
No
|
No
|
No
|
No
|
Yes, 0 violation
|
0 alert
|
25
|
ZINC000252533065
|
High
|
No
|
No
|
Yes
|
No
|
No
|
Yes
|
Yes, 0 violation
|
0 alert
|
26
|
ZINC000089917273
|
High
|
No
|
No
|
Yes
|
No
|
No
|
Yes
|
Yes, 0 violation
|
0 alert
|
27
|
ZINC000001464338
|
High
|
No
|
No
|
Yes
|
Yes
|
Yes
|
Yes
|
Yes, 0 violation
|
0 alert
|
28
|
ZINC000002687799
|
High
|
No
|
No
|
Yes
|
Yes
|
No
|
Yes
|
Yes, 0 violation
|
0 alert
|
29
|
ZINC000012628414
|
High
|
No
|
No
|
No
|
No
|
No
|
Yes
|
Yes, 0 violation
|
0 alert
|
30
|
ZINC000072477563
|
High
|
No
|
No
|
No
|
No
|
No
|
No
|
Yes, 0 violation
|
0 alert
|
The Top Hits Show Stable Binding Mode
Out of the 10 hits for each conformation, only 2 hits from 1st conformation; 1 hit from 2nd conformation; and 6 hits from the 3rd conformation passed both the MM-GBSA binding energy and ADMET evaluations. These compounds show stable binding mode over time, reflected in the reduced protein and ligand RMSD halfway through the simulation (Fig. 10).
Key Protein-ligand Interactions Are Identified
The partial agonist Δ9-THC, via hydroxyl substituent on the polycyclic ring, forms 2 H-bonds with K109 and S90 in 35% of the simulation. Similarly, the top 9 hits demonstrate similar persistent interactions with all the 3 most abundant conformations of CB2R. Other key-interacting residues are found in common to the Δ9-THC bound CB2R and the top 9 hits are tabulated (Table 3). Unlike the alkyl chain of Δ9-THC which does not engage in the persistent interaction with CB2R, the aromatic chain in all the 9 compounds forms stable interactions with residues along the CB2R pocket (Fig. 11), accounting for their higher docking and MM-GBSA binding energy scores (Table 1). The timeline of the individual interaction is shown in Figure S15. The ligand interacted with a combination of different polar and hydrophobic interactions all over the CB2R channel.
Table 3
Key-interacting residues. These are residues involved in persistent interaction between CB2R and Δ9-THC, and the top 9 hits.
S/N
|
Compound
|
F87
|
S90
|
F91
|
F94
|
F106
|
K109
|
F183
|
Ref.
|
Δ9-THC
|
Yes
|
Yes
|
Yes
|
No
|
Yes
|
Yes
|
No
|
1
|
ZINC000009762581
|
Yes
|
Yes
|
Yes
|
Yes
|
Yes
|
Yes
|
Yes
|
2
|
ZINC000096124070
|
Yes
|
Yes
|
Yes
|
No
|
Yes
|
Yes
|
Yes
|
20
|
ZINC000257304951
|
Yes
|
Yes
|
Yes
|
Yes
|
Yes
|
Yes
|
No
|
21
|
ZINC000013475970
|
No
|
No
|
Yes
|
Yes
|
No
|
No
|
Yes
|
23
|
ZINC000408739159
|
Yes
|
Yes
|
Yes
|
Yes
|
No
|
No
|
Yes
|
24
|
ZINC000013356709
|
Yes
|
Yes
|
Yes
|
Yes
|
No
|
No
|
Yes
|
26
|
ZINC000089917273
|
Yes
|
Yes
|
Yes
|
Yes
|
Yes
|
Yes
|
Yes
|
28
|
ZINC000002687799
|
Yes
|
Yes
|
Yes
|
Yes
|
Yes
|
Yes
|
Yes
|
29
|
ZINC000012628414
|
Yes
|
Yes
|
Yes
|
Yes
|
Yes
|
Yes
|
Yes
|