Docking Studies of Pyrimidine Analogues on Geranyl Transferase

Introduction Rab proteins are small GTPases involved in the regulation of vesicular traffic [1]. To be functional, they have to undergo a post-translational modification in which one or two isoprenoid geranylgeranyl groups are attached to their C termini [2]. Rab geranylgeranyltransferase (RabGGTase or GGTase type II) catalyzes this reaction. RabGGTase belongs to a family of prenyltransferases that includes farnesyltransferase (FTase) and geranylgeranyltransferase type I. These enzymes are highly conserved in sequence and structure and possess a single active site [3]. Pyrimidines are an important class of heterocyclic compounds, which possess a wide range of biological activities such as anticancer [4,5], antibacterial [6], anti-inflammatory [7], antiviral [8], antitubercular [9], antihypertensive [10] and anticonvulsant [11] activities. The presence of 4-OCH3 and 2-NO2 and 3-NO2 substitutions on pyrimidines shows significant increase in antitubercular activity [12]. The bromo substituted, 2-hydroxy phenyl substituted pyrimidines also shows good antihistaminic activity [13]. To know the possibility of binding of RabGGTase protein to pyrimidine analogues is fascinating and molecular docking is frequently used to predict the binding orientation. Docking is used to bring out new ligands for biological targets with a known 3D structure [14]. Docking studies of the compounds were performed using GLIDE (Schrödinger, LLC, Portland) [15,16].


Introduction
Rab proteins are small GTPases involved in the regulation of vesicular traffic [1]. To be functional, they have to undergo a post-translational modification in which one or two isoprenoid geranylgeranyl groups are attached to their C termini [2]. Rab geranylgeranyltransferase (RabGGTase or GGTase type II) catalyzes this reaction. RabGGTase belongs to a family of prenyltransferases that includes farnesyltransferase (FTase) and geranylgeranyltransferase type I. These enzymes are highly conserved in sequence and structure and possess a single active site [3].
To know the possibility of binding of RabGGTase protein to pyrimidine analogues is fascinating and molecular docking is frequently used to predict the binding orientation. Docking is used to bring out new ligands for biological targets with a known 3D structure [14]. Docking studies of the compounds were performed using GLIDE (Schrödinger, LLC, Portland) [15,16].

Protein structure preparation
The X-ray crystal structure of protein geranyl transferase in complex with a peptidomimetic inhibitor (PDB ID: 3HXB) obtained from the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank ( Figure 2) was used in this study. Water molecules of crystallization were removed from the complex and the protein was optimized for docking using the protein preparation and refinement utility provided by Schrödinger LLC. Partial atomic charges were assigned according to the OPLS-AA force field.

Docking protocol
All docking calculations were performed using the ''Extra Precision'' (XP) mode of GLIDE program. The binding site, for which the various energy grids were calculated and stored, is defined in terms of two concentric cubes: the bounding box, which must contain the center of any acceptable ligand pose, and the enclosing box, which must contain all ligand atoms of an acceptable pose, with a Root Mean Square Deviation (RMSD) of less than 0.5 Å and a maximum atomic displacement of less than 1.3 Å were eliminated as redundant in order to increase diversity in the retained ligand poses. The scale factor for van der Waals radii was applied to those atoms with absolute partial charges less than or equal to 0.15 (scale factor of 0.8) and 0.25 (scale factor of 1.0) electrons for ligand and protein, respectively. The max keep variable which sets the maximum number of poses generated during the initial phase of the docking calculation were set to 5000 and the keep best variable which sets the number of poses per ligand that enters the energy minimization was set to 1000. Energy minimization protocol includes dielectric constant of 4.0 and 1000 steps of conjugate gradient. Upon completion of each docking calculation, at most 100 poses per ligand were generated. The best docked structure was chosen using a GLIDE score (Gscore) function. Another scoring function used by GLIDE is E-model, which itself derived from a combination of the Gscore, Coulombic, van der Waals and the strain energy of the ligand.

Results from QikProp
The ADME properties of the designed ligands were predicted using QikProp. The compounds prepared were subjected to druglikeness filter. The criteria of the filter includes molecular weight 160-480, number of heavy atoms 20-70, lipophilicity 40-130, Number of hydrogen bond donors 4-7, Number of hydrogen bond acceptors 8-12. All the designed ligands conformed to the above mentioned criteria and they were evaluated for docking using GLIDE software.

Results from SiteMap
The active site on the protein was predicted by SiteMap ( Figure  3). The protein consisted of five active sites. The yellow, blue and red meshes represent the hydrophobic map, hydrogen bond donor, and hydrogen bond acceptor respectively.

Receptor grid generation
GLIDE receptor grid was generated to determine the size of the

QikProp analysis
QikProp efficiently evaluates pharmaceutically relevant properties for over half a million compounds per hour, making it an indispensable lead generation and lead optimization tool. Accurate prediction of Absorption, Distribution, Metabolism, Elimination (ADME) properties prior to expensive experimental procedures, such as High Throughput Screening (HTS), can eliminate unnecessary testing on compounds that will ultimately fail; ADME prediction can also be used to focus lead optimization efforts to enhance the desired properties of a given compound [15,16].

SiteMap analysis
SiteMap proven algorithm for binding site identification and evaluation can help researchers to locate binding sites with a high degree of confidence and predict the druggability of those sites. Beyond lead discovery, SiteMap assists in lead optimization by providing insight into ligand-receptor interactions so as to suggest effective strategies to modify lead compounds to enhance receptor complementarity [17].  active site (Figure 4). The most probable orientation of the ligands in the binding pocket is identified and a scoring function is used to quantify the strength of the interaction a molecule can make in a particular orientation. In order to provide better correlation between good poses and good scores, the GLIDE XP precision was favored over the standard mode.
The most straightforward method of evaluating the accuracy of a docking procedure is to determine how closely the lowest energy pose (binding conformation) predicted by the object scoring function. In the present study, Extra Precision GLIDE docking procedure was validated by removing the peptidomimetic inhibitor compound 6     from the binding site and redocking the six pyrimidines analogues into the binding site of RabGGTase. We found a very good agreement between the localization of the inhibitor upon docking and from the crystal structure of the protein. The binding affinity of the ligands with RabGGTase protein has been analyzed from the G-score, GLIDE energy and H-bonds. To study the molecular basis of interaction and affinity of binding of pyrimidine analogues to RabGGTase protein, all the ligands were docked into the active site of RabGGTase. The docking result of these ligands is given in table 2. The ranking of ligands was based on the GLIDE score. All the 6 ligands accepted poses with the receptor (3HXB). The interaction energy includes van der Waals energy, electrostatic energy, as well as intermolecular hydrogen bondings were calculated for each minimized complex ( Table 2). The difference in GLIDE score among all the 6 ligands was also very small. The docking score using GLIDE varied from -1.21 to -2.3. The GLIDE Score for Zarnestra, a standard inhibitor of farnesyltransferase, docked with RabGGTase was -3.71. This proves that pyrimidine analogues could be potential drugs for anticancer drug development.