Malaria is a parasitic disease which can be fatal if left untreated. Many classical anti malarial drugs have developed resistance in the parasite, hence, we need to identify unique biochemical processes known as New Permeability Pathways (NPPs) in order to develop novel drugs. P. falciparum uses de novo pathway to synthesize pyrimidine bases.
Dihydroorotate dehydrogenase (DHODH) is a flavin-dependent mitochondrial enzyme that catalyzes the fourth reaction in this essential pathway; coenzyme Q (CoQ) is utilized as the oxidant. PfDHODH is therefore an important drug target. This paper describes 3D QSAR and pharmacophore modelling based on derivatives of dihydrothiphenone (DHTP) inhibitors were used for building new inhibitors. A group of 38 inhibitors derived from DHTP scaffold. Biochemical assays were performed and IC50 values were obtained using 19 of these molecules. Bioactivities ranged from 6-38,500 nM. The remaining 19 were used as a test for the predicitive capabilities of the 3D QSAR pharmacophore model that was built using the HypoGen DS4 algorithm based on the data obtained from the training set of 19 molecules.
Hypo1-10 – top 10 compounds with high correlation coefficient, reduced RMS and low overall cost were selected. To predict activity of these compounds, the test set of 19 molecules were used. RMSD denotes correlation between experimental and predicted activity. Fischer’s randomization algorithm was applied to the training set’s experimental activity.
The cost value of Hypo1 was lower than the randomised compounds. It also showed 95% true correlation with training set compounds as well as lowest RMSD. It was the validated pharmacophore used for screening of 265242 compounds and the retrieval of 3529 hits from the NCI database. The hits with the best values of fit (>= 9) were docked into the virtual crystal structure of PfDHODH. Virtual docking of the DSM1 inhibitor to the molecule was used for structure based screening using Protein Preparation Wizard to optimise the structure with drug likeliness metrics, removal of water molecules (except for one essential W15 mol) and force fields.
The Glide SP docking program was used to generate every single ligand’s plausible 3D conformations and the best conformation for each hit was selected using the G-score. Initially 2150 hits were obtained but after applying ;= -5kcal/mol energy threshold and using Glide XP, 809 hits were shortlisted. Prime MM GBSA method was used for calculation of binding free energies of various conformations in the docked structure. The formula used is:G = EMM + GSGB + GSA?Gbind = Gcomplex ? (Gprotein + Gliagnd), whereEMM – Molecular Mechanics energyGSGB – surface generalised born polar solvation energyGSA – Nonpolar solvation termG – Gibbs free energy?Gbind – Binding free energy (entropy)The 235 hits with the best scores were tested virtually for protein-inhibitor interactions similar to the ones in crystal structures.
The molecules which exhibited favourable interactions with with PfDHODH were superimposed with 8 of the known inhibitors. Out of these, 235 structurally similar compounds were filtered and clustered using Cluster Ligands on the basis of Tanimoto coefficient (Tc). Factors like binding positions, diversity of conformations and availability were used for shortlisting 39 compounds. After this, the selected hits were once again superimposed with 100 known compounds and they showed a wide range of varying similarities. This proved that the compounds were structurally relatively novel. These lead molecules went through biochemical (colorimetric) assays against PfDHODH. The standard colorimetric DHODH continuous assay that monitors 2,6-dichloroindophenol (DCIP) reduction was adapted to an end-point.
The absorbance of each well was measured at 600 nm using a microplate reader. A ‘hit’ was recorded when a well had a measured absorbance greater than four standard deviations from the mean absorbance value of the no-drug DMSO control. The results were obtained as IC50 values with reference to DSM265 which had an IC50 value of 0.008 ?M.
At 10?M, the compounds NSC332161 showed inhibition of PfDHODH 65%, NSC85749 at 46% and NSC72405 at 30 %. Molecules showing less than 30% inhibition were rejected. From the prev 235 hits, another 23 compounds with two dimensional structures scaffold similar to these three were selected and screened for high activity. Lactate dehydrogenase and hypoxanthine incorporation assays were used for testing of anti-plasmodium activities for the leads. Molecular docking studies were performed on the three leads (NSC336047, NSC332161, NSC343533) and it was infered that they all have very similar docking mechanisms and interactions with the active site of PfDHODH. The structures that are involved in the binding with the active site and the binding site residues were investigated.
They were also docked against human DHODH (hDHODH) and the various interactions between each of the molecule and these two enzymes were noted. NSC336047 was found to be species selective. It showed poor inhibition against hDHODH and is selective to the amino acids L172, F188, L240 and M536 in PfDHODH that are absent in hDHODH. ECFP_4 fingerprint protocol was used for novelty testing of the lead compounds and highest, lowest and mean similarities by comparing them with known inhibitors in ChEMBL database. The three lead compounds discussed had Tc similarity values of 0.
67-0.04, 0.59-0.06 and 0.47-0.04 respectively.
It was concluded that these 3 compounds have not yet been published as antimalarial drug compounds. Cell based assays, SAR analysis and such techniques can be used in future to identify more potent and diverse PfDHODH inhibitors. On this basis, a second generation library of the most potent inhibitors is likely to lead to compounds that are effective in whole cells.