br Molecular modeling simulation study Molecular
Molecular modeling simulation study Molecular modeling study was performed through docking of the test compounds into the binding site of hDHFR enzyme using Discovery Studio 2.5 software. Computational docking is an algorithm designed to determine the suitable position and the orientation of the test compound’s pose inside the binding site; and to calculate the docking energy score (protein ligand interaction energy). In the present study, C-Docker protocol including CHARMm forcefield was used for calculating the energy scores of the enzyme and the individual ligands or the enzyme ligand complex; where the receptor is held rigid while the ligands are allowed to flex during the refinement , . In an attempt to explain the activity and gain further insight for the binding mode and the orientation of the test compounds into the ATP binding site of hDHFR enzyme, docking studies were performed. The selected docking pose was chosen based on the similarity of its binding mode to that of the lead compound MTX. The crystal structure of hDHFR in complex with MTX was obtained (PDB code: 1U70), , , . The crystal structure of of hDHFR in complex with MTX showed a hydrogen bond formed with Asn 64, Arg70, Lys68, Glu30, Ile7 and Val115; and π-interaction with Ile60. Validation of docking alogorithm was achieved, with root mean square difference (RMSD) of 1.391 Å between the top docking pose and the original crystallographic geometry. Docking of the target compounds revealed that the core scaffolds adopted volumes and orientations in the hinge region as that of MTX. The difference in docking energy scores of test compounds and amino porcine binding mode revealed that rigidity with fixed orientation of the cyclized structures 18 and 43 and their lower energy scores caused better binding mode which favor the biological activity. List of the calculated C-Docking score difference (kcal/mol) of the test compounds is showen in Table 1. Compounds 18, 19, 43–54 formed hydrogen bond between the N1-nitrogen of the pyrdazine ring with Glu30; and the carbonyl group with Trp24, Arg70 or Lys64. The thiazolopyridazine and imidazo-thiazolo-pyridazine moieties were both oriented deep in the hydrophobic pocket at the back of the ATP binding site. In addition π-cation interaction with Arg22 and π-π interaction with Phe31 residues were observed in most of the tested compounds. Orientation of the test compounds towards Arg22 and Phe31 which are not among the recognition key residues of the parent MTX, may interpret the inhibitory activity exhibited by compounds of this new series. The lowest energy-minimized structures (Fig. 5) of the most active 13 & 43, the least active 50 and MTX (IC50, 0.08 μM) were used in the subsequent modeling experiments. The 2D binding mode of 13 and 43 were shown in Fig. 6. The said compounds showed high affinity binding energy score difference of 46.12 and 46.65 towards Phe 31 residue, respectively; while Arg22 residue is linked to phenoxy moiety for 13 and imidazo[2′,1′:2,3]thiazolo-[4,5-d]pyridazine moiety for 43, in addition to a network of π-π interaction and hydrogen bonding. This pattern of binding explains the diminished activity of compound 50 (binding energy score difference of 40.66), which lack any type of binding with those amino acids. Ligand-based active site alignment study by docking inside the binding pocket is a well-known technique for structural analysis of ligand complexes . Flexible alignment comparative modeling experiment was performed (Fig. 7). Initial approach was applied to employ CharmM/MMFF94 flexible alignment automatically generated superposition with minimal user bias. There was a good alignment profile between compounds 13, 43 and MTX explaining its activity pattern. Fig. 7c clearly indicates a different alignment profile where carbonyl groups are not on the same side between 50 and MTX which is in consistency with the obtained experimental data. The surface map for the DHFR binding pocket was calculated . MTX showed to occupy the whole space lying into the groove pocket (Fig. 8). Compounds 13 and 43 active site alignment showed a pattern which resemble that of MTX, filling all the area of the active site with its bulkiness and fit into the hydrophobic pocket forming favorable binding contact. On the other hand, compound 50 was pointed out toward the surface wall of the active site and deprived of any receptor exposure clashes explaining its poor DHFR inhibitory activity. The hydrophobic distributions of the least active compound 50 showed less lipophilic moieties and hence the required lipophilicity for effective binding to DHFR is absent.