In silico designing and molecular docking of a potent analog against Staphylococcus aureus porphobilinogen synthase.

BACKGROUND: The emergence of multidrug-resistant strains of Staphylococcus aureus, there is an urgent need for the development of new antimicrobials which are narrow and pathogen specific. AIM: In this context, the present study is aimed to have a control on the staphylococcal infections by targeting the unique and essential enzyme; porphobilinogen synthase (PBGS) catalyzes the condensation of two molecules of δ-aminolevulinic acid, an essential step in the tetrapyrrole biosynthesis. Hence developing therapeutics targeting PBGS will be the promising choice to control and manage the staphylococcal infections. 4,5-dioxovalerate (DV) is known to inhibit PBGS.
MATERIALS AND METHODS: In view of this, in this study, novel dioxovalerate derivatives (DVDs) molecules were designed so as to inhibit PBGS, a potential target of S. aureus and their inhibitory activity was predicted using molecular docking studies by molecular operating environment. The 3D model of PBGS was constructed using Chlorobium vibrioform (Protein Data Bank 1W1Z) as a template by homology modeling method.
RESULTS: The built structure was close to the crystal structure with Z score - 8.97. Molecular docking of DVDs into the S. aureus PBGS active site revealed that they are showing strong interaction forming H-bonds with the active sites of K248 and R217. The ligand-receptor complex of DVD13 showed a best docking score of - 14.4555 kcal/mol among DV and all its analogs while the substrate showed docking score of - 13.0392 kcal/mol showing interactions with S199, K217 indicating that DVD13 can influence structural variations on the enzyme and thereby inhibiting the enzyme.
CONCLUSION: The substrate analog DVD13 is showing significant interactions with active site of PBGS and it may be used as a potent inhibitor to control S. aureus infections.

Staphylococcus aureus is the causative agent of many opportunistic infections in humans and animals.[1] As a human pathogen, S. aureus causes superficial, deep-skin, and soft-tissue infections, endocarditis, and bacteremia, as well as a variety of toxin-mediated diseases, including gastroenteritis, staphylococcal scalded skin syndrome, and toxic shock syndrome.[2] Among animals, from whose milk it is frequently isolated, it is the leading cause of intramammary infections in cows, with major economic repercussions.[3] A recent meta-analysis suggested that mortality due to multidrug-resistant S. aureus in the United States may exceed that from human immunodeficiency virus infections and AIDS.[4] This has resulted in a renewed interest in identifying new targets and molecules effective against multidrug-resistant strains of bacteria, and S. aureus. The need to identify such untapped potential antibiotic targets in bacterial pathogens has fostered innovative approaches for essential gene identification.[56]

In this quest porphobilinogen synthase (PBGS), also known as δ-aminolevulinic acid dehydratase (ALAD), catalyzes the first common step in the biosynthesis of the tetrapyrrole pigments (heme, chlorophyll, vitamin B12). ALAD catalyses the condensation of two δ-aminolevulinic acid (ALA) molecules to form the pyrrole porphobilinogen (PBG). Four PBG molecules are linked together by porphobilinogen deaminase and then cyclized by uroporphyrinogen synthase to form uroporphyrinogen III, which can be further modified to produce different metallo-prosthetic groups such as heme, chlorophyll and vitamin B12.[78910] ALAD catalyzes the formation of an asymmetric pyrrole from two identical substrates. Single-turnover experiments have proven that the first substrate molecule entering the active site finally forms the propionate side of the product PBG and the second molecule forms the acetate side. According to this, the two substrates are distinguished as the “P-site” and “A-site” substrates.[11] Most ALADs are dependent on divalent cations for activity and these metal ions participate in catalysis or allosteric regulation. The residues of the PBGS active site are highly conserved.[7] One of the key pathogenic factors of S. aureus is the formation of small colony variants (SCV) and this formation is normally observed in relapsed episodes of S. aureus infection and this pathogen shows SCV formation through the acquisition of heme exogenously[1213] and functioning of PBGS, regulates heme biosynthesis, therefore, this pathway is directly involved in the pathogenesis of S. aureus. In the present study is to identify a suitable inhibitor of PBGS, which can probably keep the S. aureus in normal morphology and their make this organism susceptible to conventional antibiotics.

In order to predict the interaction of compounds with the enzyme, it is necessary to have pure crystal structure and in the absence of such structures the amino acid sequence was BLAST searched in Protein Data Bank (PDB) and most close structure is chosen to construct the 3D structure using homology modeling method. In this process when S. aureus PBGS amino acid sequence was BLAST searched against PDB and we found the crystal structure from Chlorobium vibrioform (PDB ID: 1W1Z) showing the maximum identity of 56% therefore; it was chosen as template to construct the 3D structure of S. aureus PBGS. Further, in the present study, effort was made to generate the 3D structure of the PBGS structure and the binding affinity and interaction mechanism of 4,5-dioxovalerate (DV) and its analogs against PBGS was studied with molecular operating environment (MOE).

Materials and Methods

Sequence analysis and Homology modeling of S. aureus PBGS

As the structure of S. aureus PBGS is not available so far in the PDB[14] we have constructed its 3D model by homology modeling method using Modeler 9v8 tool.[15] The S. aureus PBGS protein sequence was subjected to BLASTp[16] against PDB and the crystal structure from C. vibrioform (PDB ID: 1W1Z) showing the maximum identity of 56%[17] was chosen as template. A sequence alignment file was generated in PIR format for query and template sequences using ClustalX tool.[18] Python script was written and 20 best models were generated. The model with the lowest discrete optimized protein energy (DOPE) score was selected for further analysis.

The stereo chemical quality of the model was validated by PROCHECK and ProSA web servers. Both can read the atomic coordinates of the 3D model and judge the quality of the structure. Ramachandran plot has been generated from PROCHECK[19] validation server was used to access the quality of the model by looking into the allowed and disallowed regions of the plot. A Z-score value was generated from ProSA web server that can determine the overall quality of the model and its identity nearest to native nuclear magnetic resonance/X-ray crystal structures.

Structural studies of PBGS

For the structural study of PBGS, the PyMOL software has been used. It helps in the visualization of proteins PDB's, removal of water and hetero atoms from protein PDB files and analysis of docking results. The position of alpha helix, beta sheets, interacting residues and active site has been studied upon through PyMOL.[20]

Molecular docking

Molecular docking was carried out using MOE docking software tool (MOE 2011.10). The 3D structure of PBGS was loaded into MOE software removing water molecules, hetero atoms and polar hydrogen's were added. The structures were protonated at temperature of 300K, pH 7 and a salt concentration of 0.1. Generalized born implicit solvating environment was enabled with a dielectric constant of 1 and Van der Waals forces were enabled at a cut-off value of 10 Å. Energy minimization was carried out in OPLS force field at a gradient of 0.05 to calculate the atomic coordinates of the protein that are local minima of molecular energy function and also to determine low energy conformations and to proceed for molecular dynamics simulations. The simulations were carried out in the same force field and NVT statistical ensemble was used where the temperature is held fixed to generate the trajectories. The most accurate Nose-Poincare-Anderson algorithm was enabled to solve the equation of motion during simulations. The initial temperature was set at 30K and increased to a run time temperature of 300K and the simulations were carried out for a total period of 10 ns and these stabilized conformations generated at the end of the simulations were used for molecular docking process.

Individual dockings were performed for DV and its 19 analogs against S. aureus PBGS to find out the binding modes and affinity variations. The simulations was loaded in to the MEO and the binding site defined with A197, S198, S199, F201, G202, R205, S213, D216, R217, K218, and E221. The data base containing 19 analogs was docked into the binding site using alpha triangle docking placement methodology and poses were generated by superposition of ligand atom triplets and triplets of receptor site points. Thirty docking conformations were generated for each ligand and these conformers were ranked by London dG scoring function to estimate the free energy of binding of the ligand from a given pose. The conformations thus were refined and rescored in the same force filed to remove the duplicate conformations. At the end of docking process the pose with least score was chosen from the total conformations and in each docking process the binding orientations was studied.[21]

Results

The crystal structure of S. aureus PBGS is not available therefore the structure was built using homology modeling method.[17] In order to predict the structure of S. aureus PBGS, 20 best models were generated for PBGS by using Modeler 9v8 tool. The X-ray crystallographic structure of PBGS from C. vibrioform (PDB ID: 1W1Z) was used as template which showed 56% sequence identity. The lowest DOPE score of − 36363.63281 was found with the model 18 among 20 models and it was considered for further study. The stereochemistry of the final model [Figure 1] was verified by submitting to PROCHECK validation server and Ramachandran plot showed 95.2% of the residues in most favorable region and no residues were found in the disallowed region [Figure 2]. Most appropriately the plot was found with favorable comparison with X-ray crystallographic data. The ProSA-Web evaluation of PBGS model revealed a compatible Z-score value of − 8.97 that falls in the range of native conformations of X-ray crystal structure [Figure 3].

Figure 1

The three-dimensional structure of Staphylococcus aureus porphobilinogen synthase with lowest discrete optimized protein energy score built from Modeller 9v8

Figure 2

Ramachandran plot generated by PROCHECK validation server showing the stereochemical quality of the Staphylococcus aureus porphobilinogen synthase structure

Figure 3

ProSA-web plot of porphobilinogen synthase structure showing the Z-score (−8.97) in the range of native X-ray crystallographic structure

The crystal structure of S. aureus PBGS was optimized by energy minimization. The molecular dynamics simulations of this structure have generated a total of 20,000 conformations for a period of 10 ns. The stabilized conformation was obtained at the end of the simulations is used for the docking purpose. The binding site residues were identified as A197, S198, S199, F201, G202, R205, S213, D216, R217, K218, and E221. The ligand database was constructed for all the ligands and all the structures were optimized to a gradient cut off value of 0.05 kcal/mol/Å. This database was docked into the specified binding domain and docking conformations were generated. The binding orientations and interaction mechanisms along with 19 ligand structures are shown in Supplementary Table 1 and the docking score information is shown in Table 1. The docking results indicates most of the ligands are interacting with the polar hydrophilic active site residues like lysine and arginine; some of the ligands are interacting with serine although hydrophilic active site with aromatic ring structure residues are interacting with all ligands the DVD13 was found to be showing least docking score and forming four hydrogen bonds with the active site among which two bonds are formed with K248, R217, and two bonds with E121 [Figure 4]. Due to best docking score − 14.4555 and good hydrogen bond interactions DVD13 is considered as an efficient PBGS inhibitor among all dioxovalerate derivatives (DVDs).

Supplementary Table 1

Molecular docking interaction of DVDs with S. aureus PBGS active site

Table 1

Molecular docking information of DVDs with S. aureus PBGS active site

Figure 4

The binding mode orientation of DVD13 in the porphobilinogen synthase active site

Discussion

PBGS, which is also known as ALAD, catalyzes a unique asymmetric condensation of two molecules of ALA to form the mono PBG. ALA and porphobilinogen are the biosynthetic precursors to all the tetrapyrrole pigments (e.g. porphyrin, chlorin, and corrin).[7] Tetrapyrroles such as hemes and chlorophyll are essential prosthetic groups involved in numerous electron transport chains for energy recovery in essentially all forms of life. Heme is a key molecule for S. aureus and is involved in many aspects of oxidative metabolism.[7] Crucially, heme is required for the activity of cytochromes of the electron transport chain. S. aureus is able to obtain heme either through biosynthesis or through acquisition from the host[12] also these organism acquire high amount of heme from host for the formation of SCV is the characteristic feature of multidrug-resistant strains of S. aureus which also show high resistance to vancomycin. In these iron acquisition up regulates tricarboxylic acid (TCA) cycle, peptidyl deformylase and production of δ-toxin and thereby spreading its pathogenesis.[1322] Therefore, these biosynthetic pathways of tetrapyrroles are correspondingly highly conserved, making heme biosynthesis an attractive target for antibacterial drug discovery and application.[23]

In the present study, the 3D model of PBGS was constructed using C. vibrioform (PDB 1W1Z) as a template by homology modeling method and the built structure was close to the crystal structure with Z score − 8.97. DV is known to inhibit PBGS due to its poor potency the structural analogs were designed to predict the inhibitory activity using molecular docking studies. After successful docking and binding mode analysis of all the ligands, DVD13 was found to be having an effective interaction with the active site of PBGS and was evidenced by its lowest docking score. To prove its potency of PBGS inhibition an in-depth analysis was carried out to determine the molecular interactions of the DVD13 with PBGS. The total conformations that were generated for DVD13 during docking process were analyzed in detail. Moreover, the DVD13-PBGS docking complex was subjected to 10ns molecular dynamics simulations to determine the stability of the DVD13 in the active site of PBGS [Table 2]. The ligand was found to be stable in the binding site throughout the simulation period and stable interactions were observed with the active site residues. In DVD13, four reactive centers were detected, i.e. R1, R2, N-Bridge and S-Center [Figure 5]. These reactive centers were playing a major role to form stable interactions to bind with PBGS active site. The S-center was observed to be highly rich in H-bonding pattern. This center was found to be forming the interactions with the polar residues like K195 and K248. The hydrophobicity of this center tends to interact with the aromatic residue F204 which will enhance the interaction by stacking of aromatic rings. The O-atom that is attached to the S-atom was found to be highly reactive where it formed H-bonds with the polar residues of R205, D216 and K248 and also with the hydroxy group of the residues S213 and S199. One more O-atom was observed attached to the C-atom which is exactly adjacent to S-center and is enhancing the interaction by forming H-bonds with the same residues like K248 and S199. The next predominant interaction was observed with R1 reactive center, which is formed of an aromatic ring bonded with one ketone and two hydroxyl groups. This center is containing an equal portion of polar, hydrophobic and H-bonding regions. Moreover, it is also forming arene cationic interactions with R205, R217, K218, and K248 residues. The H-bonding was observed with the residues like S198, D206, S210, D216, K218, and K248. In addition to this, the stable interactions, i.e. arene-arene interactions were also detected with F201 and F204. The aromaticity of this reactive center was more attractive to the phenylalanine residues of the active site. This reactive center is also making solvent contacts exposing out from the active site cavity in some conformations.

Table 2

The conformations of DVD13 in PBGS active site during 10 ns simulations. The trajectories of every 500 ps were shown in the each cell of the table

Figure 5

3D conformation of DVD13 showing reactive centers. The red colored cloud region indicates the H-bonding area, blue color indicates the hydrophobic region and the red color indicates polar reactive regions

The next reactive center is R2 where its interaction was observed with the polar residue R217 by means of arene cationic interaction. This region is rich in hydrophobicity that can also enhance and strengthen the complex. The N-bridge which is acting as barrier of the two reactive centers R1 and R2 was found to be forming H-bonds with the residues like S213 and R217 which are already in interaction with the remaining reactive centers.

These observations reveal the reactive moieties and the stable interactions strongly suggest DVD13 as an effective inhibitor of PBGS which probably can be deduced to design more potent inhibitors of PBGS. This heme catabolism which starts through PBGS is an essential feature in the regulation of TCA cycle in S. aureus, DVD13 a potential inhibitor of PBGS prevents iron catabolism may be useful as drug to control the S. aureus infections.

Conclusion

S. aureus is a Gram-positive pathogen cause's plethora of infections in human beings that survives in the nasopharyngeal tract as biofilms. The formation of SCV is the characteristic feature of multi drug resistant strains of S. aureus are linked to persistent and activating infections and are often auxotrophic for heme. Iron is a key nutrient for S. aureus, and soluble free iron is extremely limited in the host environment. S. aureus preferentially scavenges heme, in this quest PBGS catalyzes the first common step in the biosynthesis of the heme. In our study, we have designed a novel and potent analogs of DVD13 showed better docking score than all DVD's, therefore this DVD13 probably can act as antimicrobial agent in controlling the staphylococcal infections.