Molecular docking studies on DMDP derivatives as human DHFR inhibitors.

Molecular docking is routinely used for understanding drug‐receptor interaction in modern drug design. Here, we describe the docking of 2, 4-diamino-5-methyl-5-deazapteridine (DMDP) derivatives as inhibitors to human dihydrofolate reductase (DHFR). We docked 78 DMDP derivates collected from literature to DHFR and studied their specific interactions with DHFR. A new shape-based method, LigandFit, was used for docking DMDP derivatives into DHFR active sites. The result indicates that the molecular docking approach is reliable and produces a good correlation coefficient (r2 = 0.499) for the 73 compounds between docking score and IC50 values (Inhibitory Activity). The chloro substituted naphthyl ring of compound 63 makes significant hydrophobic contact with Leu 22, Phe 31 and Pro 61 of the DHFR active site leading to enhanced inhibition of the enzyme. The docked complexes provide better insights to design more potent DHFR inhibitors prior to their synthesis.


Background:
Folate metabolism has long been recognized as an attractive target for cancer chemotherapy because of its indispensable role in the biosynthesis of nucleic acid precursors [1]. Within folate metabolism, Dihydrofolate reductase (DHFR) which catalyzes the reduction of folate or 7, 8-dihydrofolate to tetrahydrofolate and intimately couples with thymidylate synthase has been of particular interest. The DHFR is present in all cells and is necessary for the maintenance of intracellular folate pools in a biochemically active reduced state [2]. Inhibition results in depletion of intracellular reduced folates, which are necessary for one carbon transfer reactions. One carbon transfer reactions are important for the biosynthesis of thymidylate, purine nucleotides, methionine, serine, glycine and many other compounds necessary for RNA, DNA and protein synthesis [3]. Therefore, DHFR represents an attractive target for developing antitumor agents.
Several DHFR inhibitors, as separate entities, have found clinical utility as antitumor agents [4]. The classical antifolate like methotrexate (MTX) has been used clinically for more than 50 years. Because of the frequent occurrence of tumor resistance and ineffectiveness against many solid tumors, extensive structural modifications of MTX have been reported to improve its antitumor spectrum of activity and to circumvent tumor resistance.
[5] However, none of these modified analogues showed better DHFR inhibitory or antitumor activity than MTX. In addition, they require an active transport mechanism to enter cells, which, when impaired, causes resistance. In an attempt to overcome these potential drawbacks, non classical lipophilic antifolates have been developed as antitumor agents which do not require the folate transport system(s) and enter cells via diffusion. One such group is the derivatives of 2, 4diamino-5-methyl-5-deazapteridine (DMDP) having structures similar to the trimetrexate/piritrexim class of antifolates. Due to an interest in new anticancer drugs, several DMDP inhibitors were chosen from the Southern Research Institute chemical repository for screening against human DHFR [6].
Nowadays, molecular docking approaches are routinely used in modern drug design to help understand drug-receptor interaction. It has been shown in the literature that these computational techniques can strongly support and help the design of novel, more potent inhibitors by revealing the mechanism of drug--receptor interaction. However, so far, there has been no report concerning the application of molecular docking methodology for understanding the binding of DMDP derivatives.
In this study, we have used docking studies to study the binding orientations of DMDP derivatives to human DHFR. Such studies have been carried out to understand the forms of interaction of seventy eight compounds, synthesized by Suling and colleagues [6] for the human DHFR. The results obtained from this study would be useful in both understanding the inhibitory mode of the DMDP derivatives as well as in rapidly and accurately predicting the activities of newly designed inhibitors on the basis of docking scores. These models also

Molecular docking
Molecular docking of DMDP derivatives to the active site of human DHFR was carried out using modern docking engine LigandFit available with Cerius2_4.9. (http://www.accelrys.com). This algorithm makes use of a cavity detection algorithm for detecting invaginations in the protein as potential active site regions. A shape comparison filter is combined with a Monte Carlo conformational search for generating ligand poses consistent with the active site shape. Candidate poses are minimized in the context of the active site using a gridbased method for evaluating protein-ligand interaction energies. The docking was carried out with the following non default settings in LigandFit: site partitioning in order to fully access the potential docking orientation of the active site, maximum trials variable table values to help the pseudorandom conformational analysis, and the CFF force field [8] option was used for the grid energy calculations. The flexible fitting option was selected for generation of alternative conformations on the fly, as was the diverse conformer's option to ensure the solutions generated covers a broad range of conformations with similar low-energy docking scores, and a maximum of 30 top scoring diverse ligand poses were returned for each of the compounds.

Scoring function
The docked conformations were further scored using various scoring functions available with Cerius2 [8]. The LigandFit algorithm [7] uses an internal scoring function, DockScore, to select and return dissimilar poses for each compound. DockScore is a simple force field based scoring function which estimates the energy of interaction by summing the ligand/protein interaction energy and the internal energy of the ligand. CFF force field [8] was used to resolve the van der Waals parameters for DockScore. The top DockScore pose was used for post docking scoring. The scoring was performed using a set of scoring functions as implemented in Cerius2 [8]. These included LigScore1, LigScore2, -PLP1, -PLP2, -PMF and DockScore available from the docking process. The putative 3D poses and score results were then stored as a SD file. Each docking was minimized, using DockScore, the only purely molecular mechanics based scoring function employed in this study, and this minimized pose was then presented to each of the other scoring functions, which were either knowledge based or regression based.

Protein preparation
The high-resolution (1.09 Å ) X-ray structure of human DHFR complex with SRI 9439 (PDBid code 1KMS) was imported into Cerius2 [8], and the ligand was extracted to leave a cavity. Thereafter, the docking simulations were carried out with and without cofactor NADPH and water molecules, to elucidate the role of NADPH and water molecules for the binding of DMDP derivatives.

Results and discussion:
To date, several crystal structure of human DHFR in complex with different inhibitors have been reported viz 1DHF with folate

Validation of the docking method
To ensure that the ligand orientation obtained from the docking studies were likely to represent valid and reasonable binding modes of the inhibitors, the LigandFit program docking parameters had to be first validated for the crystal structure (PDBid 1KMS). The ligand SRI-9439, in the conformation found in the crystal structure, was extracted and docked back to the corresponding binding pocket, to determine the ability of LigandFit to reproduce the orientation and position of the inhibitor observed in the crystal structure. Results of control docking showed that LigandFit determined the optimal orientation of the docked inhibitor, SRI-9439 to be close to that of the original orientation found in the crystal shown in Figure  1a.
The low RMS deviation of 0.502 Å between the docked and crystal ligand coordinates indicate very good alignment of the experimental and calculated positions especially considering the resolution of the crystal structure (1.09Å).

Interaction Modes between the DMDP derivatives and human DHFR
The binding modes of DMDP derivatives in the binding site of human DHFR were identified using intermolecular flexible docking simulations by means of LigandFit program. All the compounds in the dataset were docked into the active site of human DHFR, using the same protocol.  The binding mode of the most active compound 63 has been shown in Figure 1c. As expected, compound 63 bind to the DHFR active site in the similar conformation as other known DHFR inhibitors (SRI-9662, SRI-9439, methotrexate) which mainly bind using the pterin moiety and this moiety is presented to nicotinamide ring of cofactor NADPH. This pterin ring is involved in π-π stacking interactions with the nicotinamide ring of NADPH. This stacking interaction is very important and has been conserved in most of the DHFR's for which crystal structures have been solved with NADPH and inhibitors in ternary complex with the enzyme. The chloro substituted naphthyl ring of compound 63 makes significant hydrophobic contact with Leu 22, Phe 31 and Pro 61 of the DHFR active site leading to enhanced inhibition of the enzyme when compared with compound 29 (least active) where chloro substituted naphthyl ring is substituted with phenyl ring with methoxy substitution at 2 and 5 positions leading to decreased hydrophobicity of the compound and hence low amount of inhibition as shown in Figure 1d. The two amino group of the pterin ring makes strong hydrogen bond with main chain oxygen atom of Ile 7 and Val 115 and side chain oxygen atom of Glu 30. These particular interactions play a very important role in DHFR inhibition and need to be present for good inhibition by the inhibitors. Moreover, any bulkier substitution at R1 position of the DMDP derivatives may lead to steric clashes with Phe 34 and the cofactor NADPH and that is why compound 29, 45, 50, 51 are not very much active, whereas compounds with methyl substitution are more active as in case of compound 63, is shown in Figure 1c.

Correlation between docking scores and inhibitory activity
The predicted inhibitory activity of DMDP derivatives as inhibitors on the basis of dock score is listed in  Figure 2. Activity can be best explained for rest of the compounds using the Equation given in supplementary material.

Conclusion:
In this work, molecular docking studies were carried out to explore the binding mechanism of DMDP derivatives to the human DHFR enzyme to enable the design of new DMDPbased human DHFR inhibitors. Both the binding conformation of DMDP and their binding free energies were predicted by molecular docking. The binding free energies of these compounds to human DHFR were found to have a good correlation with the experimental inhibitory activities. The results provide insight into the structural requirement for the activity of this class inhibitor and the most favorable binding mode of the top -ranking compounds will be useful in designing new DMDP derivatives as human DHFR inhibitors.