Computer Aided Drug Designing of Novel Inhibitors against Pfama-1

The invasion of Plasmodium in RBC is the most important pathogenic step. Further proliferation is stopped if any drug or antibody can inhibit at this stage. Among two major invasive surface proteins PfRH5 and AMA-1, only multiple AMA-1 structures viz. 3ZWZ, 2Q8B, 2Z8V, 3SRJ are available at time of study which was selected to obtain information about the common ligand-bound interface residues. Three active sites were created and the amino acid properties provided information that some of these are polymorphic and some are hydrophobic and conserved. Docking was done using the ZINC library on the Web server. Interactions of H-bond of the crystal structure and docking results revealed that ASN223 and ILE225 amino acids were the common H-linkage formation in all structure. ILE225 is a polymorph that cannot be considered as an interaction, whereas ASN223 is a very important polar interacting residue. However, from the docking results, only the compounds interacting with ASN223 were analyzed. ZINC80342995 and ZINC95042198 have been selected as active compounds. Both have properties in the range of the ideal drug according to ADME/T properties. However, in vitro analysis will be needed to analyze the actual potential of these candidates to prevent invasion.


Mod Appro Drug Des
core topology called as PAN domain or Apple fold consisting fivestrand β sheet flanked by single α helix at one side and three stranded sheet at other side [19]. AMA-1 form complex with RON i.e. Rhoptry Neck Protein releases from the Rhoptry secretary organelle present in parasite [15]. It is inserted into the host plasma membrane (erythrocyte membrane) and form a complex of RON2/4/5/8 [20]. RON protein family of parasite is highly conserved. This complex contains RON2 (assumed to contain 3 hydrophobic helices), RON5 (contain only one predicted hydrophobic helix) and RON4, RON8 (both appear to be soluble proteins) [21].
In present study, AMA-1 structures viz., 3ZWZ, 2Q8B, 2Z8V, 3SRJ were selected to obtain the information of common interface residues. Using these residues, three active sites were generated. Properties of amino acids gave the information that some of amino acids are polymorphic and some are conserved hydrophobic. Docking was carried out with a ZINC library on a web server. H-bond interactions of crystal structure and docking results were analyzed.

Materials and Methods
Retrieval and study of AMA-1 crystal structure AMA-1 crystal structures bound with different peptides were retrieved from RCSB PDB (http://www.rcsb.org/). PDB ID of selected structures were 3SRJ, 3ZWZ, 2Z8V, and 2Q8B bound with inhibitory peptide R1, small peptide of RON2 erythrocyte receptor, IgNAR antibody and growth inhibitory antibody, respectively [19,22,23]. Protein structure and their amino acid interactions were analyzed by Discovery Studio Visualizer 3.0 (Accelrys Inc., San Diego, CA, USA) [24]. Rama chandran plot of all crystal structures were generated using RAMPAGE (University of Cambridge, Dept. of Biochemistry).

Selection of common interface residues
All bound structures contain interface residues which were found by Inter Pro Surf server (http://curie.utmb.edu/prosurf. html/) [25]. A, A, A and B, A chain of AMA-1 from 2Q8B, 2Z8V, 3SRJ and 3ZWZ were submitted for analysis of interface residues. All resulted residues of four structures were compared and common amino acids were selected using Discovery Studio Visualizer 3.0 (http://accelrys.com/).

Active site generation from interface residues
These selected residues gave the buried pockets where all the peptides were interacting. Active site residues were covering larger area so it was split into three small active sites covering different areas.

Docking studies
Generated active sites were used further for docking. Different parameters of active site were set according to auto dock vina ( Table 1). For that AMA-1 from 3ZWZ PDB structure was selected. Docking was done by idock, a multithreaded virtual screening tool (http://istar.cse.cuhk.edu.hk/idock) which is linked with the ZINC databases [26]. Filtration of ZINC database were done using Lipinski's rule of five and other parameters were set according to ideal characteristics of drugs which act on blood stages ( Table  2). The results obtained after docking were visualized in an iview (interactive WebGL visualize for protein-ligand complex) [27]. Size-X 10 10 10 Size-Y 10 10 10 Size-Z 10 10 10

Selection of hits
Hits were selected on the bases of their interaction with specific hydrophobic, conserved, non polymorphic amino acids from the given residues and forming maximum bonds. For that, residues characteristics were studied from literatures. Comparison of H-bond formation in four crystal structures and docking result was done which were also used in selection on specific amino acids. Mod Appro Drug Des ADME/T properties prediction ADME/T predictions of selected compounds were carried out using admet SAR (http://admetexp.org/) which is a user friendly interface [28]. Compounds, falling in acceptable range, were selected.

Results and Discussion
Study of AMA-1 crystal structure Among the four crystal structures of AMA-1, Ramachandran plot of 3ZWZ was created which is shown in Figure 1. It shows 0% amino acids are present in outer region of a plot while 2% are present in allowed region. Remaining residues are present in favored region. Interface residues of different structures obtained from Inter Pro Surf tool [25] were listed in excel sheet for comparison. About 12 common interacting residues present in all the structures are shown in Table 3 with its position number. Some structures are also having two chains of AMA-1 and one or two interacting amino acid might change but both were considered. This information will further useful for analysis of binding pattern. Individual crystal structures are also having numbers of other interacting amino acids [29] other than listed in Table 3. These common residues (labeled in blue color) were shown in Figure 2. Different residues were forming loops, helix, turns and sheets (Table 3 and Figure 1,2).

Active site generation and selection
Interface residues, act as an active sites [30], were split into 3 different active sites. Among all these residual active site, 1 st active site which contains PHE183, THR186, GLU187, PRO188, MET190 amino acids were forming the loop. It was proven that loop may change the shape and their positions as well as they do not play a role in protein stability [31]. There might be possibility that same compound binds with one or more active residues. Other two active sites were also having the loop forming amino acids but they even contain amino acids which were involve in helix, turns and sheets surrounding it. These sites were nearer to each other so there might be possibility that compounds interacting with 2 nd active site may also interact with amino acids present in 3 rd active site. Therefore it should be determined that these interactions are favorable of not.

Docking results and ADME/T studies
The docking result, obtained from idock, was saved in a excel sheet which contain the information required for binding analysis. idock gave 17,746,988 numbers of compounds from the ZINC database after filtrations. If the library generated in idock exceed 1,00,000 compound limit than it will take randomized 1,00,000 compound from that library. So, here docking was carried out with randomly selected 1,00,000 compounds.
The hydrogen bonding and their interactions was visualized in iview [27]. The hydrogen bonds forming amino acids of crystal structures with the peptides were shown in Figure 3 where hydrogen bonds highlighted in green colored dotted line. This analysis gave the information about amino acids and exact atoms of amino acids which play a role in H-bond formation [32].
Similar analysis was performed for all docking results. The top 20 interacting compounds were taken in consideration and their interacting amino acid: atom information was combined in Active Site (log) 1, 2, 3 in a excel sheet. Same information of crystal structures were also taken from Figure 3 in excel sheet. Here, Table 4 is showing information of amino acids participating in the hydrogen bond formation and their position in known structure of AMA-1. If any similar amino acids or atoms interact in every structure and active site, they were highlighted in same color (Table  4 and Figure 3).  From this interaction analysis, two amino acids were found commonly interacting in four structures and reciprocated in docking results. ASN223 (OD1, ND2-are oxygen and nitrogen present at delta position respectively) and ILE225 (N) amino acids were forming H-bonds with the peptides and docked compounds. Apart from these, other amino acids were also forming H-bonds. From the literature review the properties of different amino acids play role in the interaction is summarized in (Table 5). It was proven that ILE225 is polymorphic and due to this nature it cannot be considered further as mutation at this residue abrogate the binding with peptides Coley et al. [19]. Second amino acid ASN223 reported in making very important polar interaction [22]. Though it does not fall in a hydrophobic conserved amino acids however does not change during every cell cycle. Compounds which interact with ANS223 that also interact with side amino acids and block that groove so that RBC receptor or other peptides cannot bind with it. Therefore compounds having top docking scores and specifically interacting with ASN223 amino acid were selected. Two compounds ZINC80342995 and ZINC95042198 were predicted as good effective hits. Structures of these compounds are shown in Figure 4 and their interactions in docking with AMA-1 are shown in (Figure 4,5). These two compounds were interacting with ASN223 and other amino acids. All the H-bonds are shown in sky blue color. More H-bonds form more stronger interaction and it also require more energy for removal of bound compound from the active site. Therefore it will be favorable if any compound interacts with more H-bonds. Here, ZINC80342995 and ZINC95042198 are forming 9 6/7

Mod Appro Drug Des
and 4 H-bonds respectively. Therefore, ZINC80342995 could be a better inhibitor. Both the structures are satisfying the Lipinski's rule of five and also the polar and non-polar dissovation, tPSA (topological polar surface area) range. The ADME/T properties of both compounds are given in Table 6 which shows that it is not harmful for human consumption. Therefore, we can conclude that both compounds are having ideal drug properties. But all the results were predicted by the software which further needed to confirm by in vitro and then in vivo experiments to prove its activity (Table 6).