Evolutionary adaptation of DHFR via expression of enzyme isoforms with various binding properties and dynamics behavior: a bioinformatics and computational study

We compared the binding properties and dynamics of three experimentally reviewed isoforms of human dihydrofolate reductase (DHFR). The cytoplasmic variants including isoforms1 and 2 (iso1 and iso2) are produced by alternative splicing; while the mitochondrial form is located in the mitochondria. The iso1 as the canonical sequence contains 187 residues, and iso2 differs from the iso1, where it has 1–52 residues missing at the N-terminus of canonical sequence. Here, the structural models of the iso2 and mitochondrial forms were constructed by the MODELLER program using the crystal structure of the iso1 as the template. Bioinformatics analysis on ligand-bearing structures demonstrates that mitochondrial variant forms more stable complex with ligands compared with iso1 and 2, indicating their different binding properties. The root mean square fluctuation (RMSF) data suggest that C-terminus of iso1 contains two representative highly flexible fragments, while iso2 contains a highly flexible fragment at N-terminus end. Interestingly, both ends of mitochondrial variant have a degree of rigidity. Finally, the observation of differences in structural dynamics and binding properties predicts that the simultaneous existence of enzyme isoforms is a way to increase the speed of the enzyme maneuver in response to various environmental conditions. This prediction needs to be tested experimentally.

The Complementarity Principle—One More Step towards Analytical Docking on the Example of Dihydrofolate Reductase Complexes

New approaches to assessing the “enzyme–ligand” complementarity, taking into account hydrogens, have been proposed. The approaches are based on the calculation of three-dimensional maps of the electron density of the receptor–ligand complexes. The action of complementarity factors, first proposed in this article, has been demonstrated on complexes of human dihydrofolate reductase (DHFR) with ligands. We found that high complementarity is ensured by the formation of the most effective intermolecular contacts, which are provided due to predominantly paired atomic–atomic interactions, while interactions of the bifurcate and more disoriented type are minimized. An analytical docking algorithm based on the proposed receptor–ligand complementarity factors is proposed.

Structural Insights of Three 2,4-Disubstituted Dihydropyrimidine-5-carbonitriles as Potential Dihydrofolate Reductase Inhibitors

In this report, we describe the structural characterization of three 2,4-disubstituted-dihydropyrimidine-5-carbonitrile derivatives, namely 2-{[(4-nitrophenyl)methyl]sulfanyl}-6-oxo-4-propyl-1,6-dihydropyrimidine-5-carbonitrile 1, 4-(2-methylpropyl)-2-{[(4-nitrophenyl)methyl]sulfanyl}-6-oxo-1,6-dihydropyrimidine-5-carbonitrile 2, and 2-[(2-ethoxyethyl)sulfanyl]-6-oxo-4-phenyl-1,6-dihydropyrimidine-5-carbonitrile monohydrate 3. An X-ray diffraction analysis revealed that these compounds were crystallized in the centrosymmetric space groups and adopt an L-shaped conformation. One of the compounds (3) crystallized with a water molecule. A cyclic motif (R22(8)) mediated by N–H···O hydrogen bond was formed in compounds 1 and 2, whereas the corresponding motif was not favorable, due to the water molecule, in compound 3. The crystal packing of these compounds was analyzed based on energy frameworks performed at the B3LYP/6-31G(d,p) level of theory. Various inter-contacts were characterized using the Hirshfeld surface and its associated 2D-fingerprint plots. Furthermore, a molecular docking simulation was carried out to assess the inhibitory potential of the title compounds against the human dihydrofolate reductase (DHFR) enzyme.

In Silico Study Identified Methotrexate Analog as Potential Inhibitor of Drug Resistant Human Dihydrofolate Reductase for Cancer Therapeutics

Drug resistance is a core issue in cancer chemotherapy. A known folate antagonist, methotrexate (MTX) inhibits human dihydrofolate reductase (hDHFR), the enzyme responsible for the catalysis of 7,8-dihydrofolate reduction to 5,6,7,8-tetrahydrofolate, in biosynthesis and cell proliferation. Structural change in the DHFR enzyme is a significant cause of resistance and the subsequent loss of MTX. In the current study, wild type hDHFR and double mutant (engineered variant) F31R/Q35E (PDB ID: 3EIG) were subject to computational study. Structure-based pharmacophore modeling was carried out for wild type (WT) and mutant (MT) (variant F31R/Q35E) hDHFR structures by generating ten models for each. Two pharmacophore models, WT-pharma and MT-pharma, were selected for further computations, and showed excellent ROC curve quality. Additionally, the selected pharmacophore models were validated by the Guner-Henry decoy test method, which yielded high goodness of fit for WT-hDHFR and MT-hDHFR. Using a SMILES string of MTX in ZINC15 with the selections of ‘clean’, in vitro and in vivo options, 32 MTX-analogs were obtained. Eight analogs were filtered out due to their drug-like properties by applying absorption, distribution, metabolism, excretion, and toxicity (ADMET) assessment tests and Lipinski’s Rule of five. WT-pharma and MT-pharma were further employed as a 3D query in virtual screening with drug-like MTX analogs. Subsequently, seven screening hits along with a reference compound (MTX) were subjected to molecular docking in the active site of WT- and MT-hDHFR. Through a clustering analysis and examination of protein-ligand interactions, one compound was found with a ChemPLP fitness score greater than that of MTX (reference compound). Finally, a simulation of molecular dynamics (MD) identified an MTX analog which exhibited strong affinity for WT- and MT-hDHFR, with stable RMSD, hydrogen bonds (H-bonds) in the binding site and the lowest MM/PBSA binding free energy. In conclusion, we report on an MTX analog which is capable of inhibiting hDHFR in wild type form, as well as in cases where the enzyme acquires resistance to drugs during chemotherapy treatment.