Development of Xanthine Based Inhibitors Targeting
3.3. Results and Discussion
3.3.5. Comparative interaction study of the best compounds from set-1 and set-2 with various members of PDE superfamily
3.3.5. Comparative interaction study of the best compounds from set-1
Whereas selected set-2 compound formed only one H-bond with GLN453. The second H bond (which was formed in case of set-1 compounds) broke as the compound was pulled away. As seen from figure 3.7, the –NH group of N7 position was pulled away from the GLN453 side chain. This condition arose due to strong interaction established by polar aromatic fragment at N3 position of compound 40 in the histidine rich site of PDE9A active site. Hence, due to presence of aromatic fragments, set-2 compounds formed better overall interaction than set-1 compounds. However, in terms of better selectivity of the compound/inhibitor by interaction with GLN453 (the specificity deciding amino acid), set-1 compounds showed better interaction with PDE9A (they formed two hydrogen bond as seen from figure 3.5).
Comparative interaction analysis with cGMP specific phosphodiesterase - PDE5A
Both PDE9A and PDE5A are cGMP specific proteins but their structures are very different from each other. PDE9A forms dimer in natural state whereas, PDE5A does not form dimer. Because both have specificity towards cGMP, it was very important to modify inhibitors in such a way that the inhibitor will be specific towards PDE9A. The best compounds from both set-1 (compound 34) and set-2 (compound 40) showed significant difference in the interaction pattern with PDE9A and PDE5A. To inhibit the protein, the compound should be able to enter into the active site. Both compound 34 and compound 40 completely entered into the active site pocket of PDE9A whereas, in case of PDE5A, they restricted at the mouth of the active site pocket of PDE5A as seen from figure 3.9. The reason behind this might be either its bigger size than the active site pocket or repulsion towards the compound in the active site of PDE5A. The compound was restricted at the entrance of the active site though, the compound interacted by one
binding pattern of compound between two cGMP specific PDEs (PDE9A and PDE5A) which led to confirm the specificity of compounds towards PDE9A. Figure 3.9 depicts the surface view and binding patterns of compound 34 and compound 40 bound PDE5A.
Figure 3.9 Surface view of PDE5A-compound 34 complex (left) and compound 40 complex (right) in PDE5A
Table 3.4 Comparative analysis of docking of compound 34 and 40 with various members of PDE superfamily
Type Specificity Best compound (compound 34) from set-1 Best compound (Compound 40) from set-2 LFEB
(kcal/mol)
NCLC No. of clusters
Interacting Residues
H bonds
LFEB (kcal/mol)
No. of clusters
NCLC Interacting Residues
H- bonds
PDE 9A cGMP -9.40 88 3 GLN453, TYR424,
PHE456
3 -12.03 90 2 HIS252, THR363,
GLN453
3
PDE5A cGMP -8.43 84 2 LYS812 1 -7.18 40 8 LYS812 1
PDE4D cAMP +13.46 - - No H-bond 0 +19.96 - - No H-bond 0
PDE7A cAMP -8.87 98 2 GLN413 1 -10.98 GLN413, HIS212 2
PDE8A cAMP -5.22 Not entered into the active site 0 -6.98 Not entered into the active site 0
PDE1B dual specific -7.16 40 9 GLN421 -9.04 61 10 HIS223, GLU293 2
PDE3B dual specific +23.21 - - No H-bonding 0 +43.09 - - No H-bonding 0
PDE2A dual specific -8.61 42 3 GLN859 1 -10.27 22 9 ASP808
PDE10A dual specific -7.55 79 3 TYR524 1 -10.17 22 11 GLU592 1
NCLC=Number of conformation in largest cluster, LFEB=Lowest free energy of binding, H-bond=Hydrogen bond
Comparative interaction analysis with cAMP specific phosphodiesterase PDE4D, PDE7A and PDE8A
The phosphodiesterase enzymes which show specificity toward the substrate cAMP are commonly known as cAMP specific phosphodiesterase. PDE4D, PDE7A and PDE8A fall into this category. Among all these, PDE8A active site is very similar to PDE9A due to the presence of TYR748. TYR is present at the entrance of active site pocket of both PDE9A (TYR424) and PDE8A (TYR748), whereas, in other PDEs, the corresponding position is occupied by phenylalanine (Huai et al., 2004; Singh and Patra, 2014). Therefore, the binding pattern of most of the compounds was similar in both PDE9A and PDE8A. Interestingly, presence of GLU406 in PDE9A fixed the orientation of amide of GLN453 side chain by the formation of intermolecular hydrogen bond.
GLU406 also created difference in the binding pattern in both PDE8A and PDE9A.
However, compound 38-42 showed significant difference in the binding pattern of ligands in the active site of PDE9A and PDE8A. In case of PDE8A, compound 38-42 was unable to enter into the active site pocket of the enzyme and was binding somewhere else on the surface of the protein as shown in figure 3.10. The main reason behind this significant difference in the binding pattern between PDE8A and PDE9A was the substitution with alkyl group at the N1 position of compounds 38-42 of set-2. The compounds having carbonylated aliphatic fragment were unable to make such difference in the interaction pattern in the active site of PDE8A and PDE9A. In Compounds 38-42, interesting difference was encountered in the binding pattern of PDE8A and PDE9A because of the presence of alkyl group at N1 position. But only substitution with alkyl group at N1 position was not sufficient to determine the difference in binding pattern
substituent (phenyl ring with alkyl substitution at meta-position) at C8 position were together responsible for creating such difference in the binding pattern of the compound 38-42 of set-2 with PDE9A and PDE8A. Among all selected compounds, compound 40 showed best interaction towards PDE9A maintaining the difference in lowest free energy of binding of 5.05 kcal/mol between PDE9A (-12.03 kcal/mol) and PDE8A (-6.98 kcal/mol). Figure 3.10 illustrates the surface view of compound 40-PDE8A complex.
Surprisingly, compound 34 and other derivatives of set-1 also showed similar significant difference in binding pattern of PDE9A and PDE8A. This affirmed the fact that N3
modification created little impact in selectivity between PDE9A and PDE8A.
Figure 3.10 Interaction of Compound 40 with PDE8A - inhibitor bound surface view (left) and empty active site view (right)
With PDE4D, most of set-1 compounds and compound 38-42 from set-2 were unable to interact. In PDE7A, compound 34 and compound 40 interacted with lowest free energy of binding -8.87 and -10.98 kcal/mol, respectively. In PDE7A, compound 34 formed one hydrogen bond with GLN413 whereas, compound 40 formed two H-bonds with GLN413
and HIS212. These compounds were interacting with PDE7A but the strength of interaction was comparatively lower than with PDE9A.
Comparative interaction analysis with dual-specific phosphodiesterases - PDE1B, PDE2A, PDE3B and PDE10A
Some phosphodiesterases such as PDE1B, PDE2A, PDE3B and PDE10A show affinity for both substrates-cAMP and cGMP. As per many reports, PDE1B has adverse side effects on PDE9A inhibition. This is because of its abundance in the brain (Andreeva et al., 2001). To improve selectivity towards PDE9A, it was necessary to make difference in the binding pattern and binding energy between PDE9A and PDE1B.
Thus, along with higher specificity towards PDE9A it was important to reduce the side effects of PDE1B. Reducing side effects of PDE1B during inhibitor designing for PDE9A will be an important improvement while developing specific inhibitors for treatment of neurodegenerative diseases (Meng et al., 2012). Hence, compounds were modified in such a way that observable difference would be created. Both Compound 34 and Compound 40 showed noteworthy differences in free energy of binding, H-bond interaction, hydrophobic interaction and van der Waals interactions between PDE9A and PDE1B. In comparison to PDE9A, PDE1B formed lesser contacts with selected compounds of both sets. Compound 34 formed one hydrogen bond with GLN421 with lowest free energy of binding -7.16 kcal/mol whereas, compound 40 formed two H- bonds with HIS223 and GLU293 with lowest free energy of binding of -9.04 kcal/mol.
Hence, with significant difference in the level of interaction with both PDE9A and PDE1, compound 34 and compound 40 might be able to reduce the side effects of PDE1B.