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Journal of Biomolecular Structure & Dynamics, ISSN 0739-1102 Volume 28, Issue Number 2, (2010) ©Adenine Press (2010)

*Phone: +91 52 2653 3982 +91 98 3907 5970 +91 9208042135Fax: +91 52 2232 4666E-mail: ashoksharma@cimap.res.in., amangenomics@gmail.com

Amandeep Kaur Kahlon* Sudeep Roy Ashok Sharma

Biotechnology Division, Central Institute

of Medicinal and Aromatic Plants

(Council of Scientific & Industrial

Research) P.O. CIMAP, Kukrail Picnic

Spot Road, Lucknow-226015,

Uttar Pradesh, India

Molecular Docking Studies to Map the Binding Site of Squalene Synthase Inhibitors on Dehydrosqualene

Synthase of Staphylococcus Aureus

http://www.jbsdonline.com

Abstract

Dehydrosqualene synthase of Staphylococcus aureus is involved in the synthesis of golden carotenoid pigment staphyloxanthin. This pigment of S. aureus provides the antioxidant property to this bacterium to survive inside the host cell. Dehydrosqualene synthase (CrtM) is having structural similarity with the human squalene synthase enzyme which is involved in the cholesterol synthesis pathway in humans (Liu et al., 2008). Cholesterol lowering drugs were found to have inhibitory effect on dehydrosqualene synthase enzyme of S. aureus. The present study attempts to focus on squalene synthase inhibitors, lapaquistat acetate and squalestatins reported as cholesterol lowering agents in vitro and in vivo but not studied in context to dehydrosqualene synthase of S. aureus. Mode of binding of lapaquistat acetate and squalestatin analogs on dehydrosqualene synthase (CrtM) enzyme of S. aureus was identified by performing docking analysis with Scigress Explorer Ultra 7.7 docking soft-ware. Based on the molecular docking analysis, it was found that the His18, Arg45, Asp48, Asp52, Tyr129, Gln165, Asn168 and Asp172 residues interacted with comparatively high frequency with the inhibitors studied. Comparative docking study with Discovery studio 2.0 also confirmed the involvement of these residues of dehydrosqualene synthase enzyme with the inhibitors studied. This further confirms the importance of these residues in the enzyme function. In silico ADMET analysis was done to predict the ADMET properties of the stan-dard drugs and test compounds. This might provide insights to develop new drugs to target the virulence factor, dehydrosqualene synthase of S. aureus.

Key words: Staphyloxanthin; Dehydrosqualene synthase; Squalene synthase inhibitors; Scigress Explorer Ultra 7.7; Docking, Virulence factor; Discovery studio 2.0.

Introduction

Development of drug resistance to all major classes of antibiotics by bacterial pathogens is well known. Recently, development of drug resistant strains of Staphylococcus aureus has initiated a need for search of new drug targets. Attempts were made to target the golden carotenoid pigment, staphyloxanthin of S. aureus (1). This pigment functions as a virulence factor for S. aureus. Staphyloxanthin pigment acts as an antioxidant and protects the S. aureus against oxidative stress due to host immune defense by reactive oxygen species and neu-trophils (2-4). This pigment biosynthetic pathway proceeds through the head to head condensation of two molecules of farnesyl diphosphate to synthesize the C30 hydrocarbon dehydrosqualene by the dehydrosqualene synthase (CrtM) of S. aureus (1, 5). These early steps are same as eukaryotic sterol biosynthetic

Abbreviations: SQS-Squalene synthase; PMF-Potential mean force; ADMET-Absorption, Distribution, Metabolism, Excretion, Toxicity

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pathway leading to cholesterol synthesis. Liu and coworkers, focussed on the S. aureus enzyme CrtM, dehydrosqualene synthase that abstracts a proton to yield the central olefin in dehydrosqualene. Further enzymatic steps through this precursor molecule create the golden color in the final product staphyloxan-thin (6). However, the eukaryotic squalene synthase uses an NADPH dependent reduction to quench an allylic cation and yield squalene (5-8). This squalene acts as a precursor molecule in eukaryotes for the biosynthesis of cholesterol by squalene synthase. The pathways diverge at presqualene diphosphate, a com-mon intermediate for the biosynthesis of dehydrosqualene by dehydrosqualene synthase of S. aureus and squalene synthesis by human squalene synthase. Thus, it was hypothesized that S. aureus dehydrosqualene synthase (CrtM) and human squalene synthase (SQS) might possess structural similarities but only modest sequence homology (30% identity, 36% similarity) was found by clustal amino acid alignment (1). However, structural similarity between S. aureus dehy-drosqualene synthase and human squalene synthase (SQS) raised the possibilty that human SQS inhibitors developed as potential cholesterol-lowering drugs might also be active against dehydrosqualene synthase (1). Recent studies have shown that the SQS inhibitors previously tested for cholesterol-lowering activity in humans, blocked the S. aureus pigment biosynthesis in vitro (1, 9-10). This resulted in the formation of colourless bacteria, which become more susceptible to killing by human blood due to innate immune defense mechanism in a mouse infection model (1, 4).

In the present study, we have used the human SQS inhibitor, lapaquistat acetate and squalestatin analogues reported as cholesterol lowering agents in vitro and in vivo (11-16) for the docking study, to identify the binding site within the CrtM, dehydrosqualene synthase of S. aureus. Molecular dynamics, molecular simulation such as docking and QSAR computational approaches, have been widely applied to study the receptor-ligand relationship and are frequently used in the drug discovery process (17-29). In an attempt to find out the active site of dehydrosqualene syn-thase (CrtM) of S. aureus, lapaquistat acetate and squalestatin analogues were used for molecular docking analysis. Bisphosphonate derivatives reported by Liu and coworkers were used for molecular docking study to prepare the standard data set. There after we performed molecular docking study by using lapaquistat acetate and squalestatin analogues, on dehydrosqualene synthase (CrtM) of S. aureus which is responsible for the synthesis of golden color pigment, staphyloxanthin. In the end molecular docking analysis results of standard dataset were compared with the test compounds used in this study. Furthermore, in silico ADMET analysis was done by DS 2.0, accelrys ADMET descriptors module for screening the test compounds for the ADMET properties (33).

Materials and Methods

Crystal Structures of S. aureus Dehydrosqualene Synthase (CrtM) Enzyme

The crystal structures of S. aureus dehydrosqualene synthase (CrtM) (PDB-ID: 2ZCO, 2ZCP, 2ZCQ, 2ZCR & 2ZCS) was retrieved from protein data bank (30) and these target proteins were used for molecular docking study using Scigress Explorer Ultra 7.7. Before docking all these protein crystal structures were cleaned by removing the heteroatoms such as ligands, ions, water molecules, etc. H-atoms were added to these target proteins for correct ionization and tautomeric states of amino acid residues.

Known Inhibitors for Standard Dataset

The 3D structures of known human SQS inhibitors, phosphonosulfonate deriva-tives and sulfur containing farnesyl analogs were retrieved from S. aureus dehy-drosqualene synthase crystal structures co-crystallized with these inhibitors

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in protein data bank. Chemical structures of these known human SQS inhibi-tors namely, BPH-652, BPH-698 and BPH-700 were found to be bound with S. aureus dehydrosqualene synthase (CrtM) enzyme in the protein data bank with PDB-ID: 2ZCQ, 2ZCR and 2ZCS, respectively. The 3D structure of farne-syl thiopyrophosphate (farnesyl analog) was obtained from crystal structure of S. aureus dehydrosqualene synthase (CrtM) enzyme PDB-ID: 2ZCP. All these four ligands were used as known SQS inhibitors to prepare the standard data set by molecular docking studies against S. aureus dehydrosqualene synthase, CrtM (PDB-ID: 2ZCO, 2ZCP, 2ZCQ, 2ZCR and 2ZCS). The active site analysis was done by selecting neighbors within 3 Å around chosen ligand after molecu-lar docking following Lamarckian GA algorithm in Scigress Explorer Ultra 7.7 software (32).

Selection of Docking Molecules as Potent SQS Inhibitors

(a) Lapaquistat acetate. 3D chemical structure of this molecule (Figure 1A) was retrieved from pubchem substance database (31) with substance ID: 47208260 and compound ID: 9874248, submitted source: KEGG (D06609). Lapaquistat acetate (TAK-475) is a cholesterol-lowering drug, which inhibit squalene synthase which is further downstream in the synthesis of cholesterol (14). 3D structure of this molecule was downloaded in SDF file format (.sdf) and then saved in chemical sample file format (.csf) in Scigress Explorer Ultra 7.7 software (32).

(b) Squalestatin analogues. 3D chemical structures of six squalestatin analogues (Figure 1B (i-vi)), squalestatin analogs 2, 15, 20, 25, 32 and 36 were downloaded from pubchem substance database (31) in 3D SDF file format and then saved into chemical sample file format (.csf) in Scigress Explorer Ultra 7.7 software. Squalestatin analogues were derived from squalestatin 1or zaragozic acid A, with a known squalene synthase inhibitory activity in vitro (13, 15). Zaragozic acid was found to have potent squalene synthase activity that made its use as cholesterol lowering agents (15). The source of these molecules was found to be unidentified sterile fungal culture Sporormiella intermedia and Leptodontium elatius, respectively (15).

Both of these test compounds were prepared for the docking study by assigning these molecules as ligands for the study in the group atoms option in Scigress Explorer 7.7 workspace and then saved into chemical sample file format.

Scigress Explorer Ultra 7.7 Docking Software

Molecular docking was performed in the project leader of Scigress Explorer Ultra 7.7 software (32). In this docking process, we kept the target protein as rigid and ligands were kept flexible. The Scigress Docking software with FastDock com-pute engine followed the Lamarckian Genetic Algorithm (LGA) similar to Aut-oDock to search for the best conformation with reference to minimum energy in terms of PMF score (potential mean force) in (Kcal/mol) and hydrogen bonding. The docked complexes were visualized after docking in the workspace module of Scigress Explorer Ultra 7.7 software (32).

Comparative Docking Using Discovery Studio 2.0

Molecular docking analysis was performed using the Ligand Fit module of DS 2.0 for comparing the results of docking obtained with Scigress Explorer Ultra 7.7 docking software. After docking the docked complexes were visualized in the DS Visualizer (33).

In Silico ADMET Analysis

Both the standard drugs and test compounds were screened for ADMET properties using the ADMET Descriptors for in silico screening in DS 2.0 (33). The ADMET properties such as absorption, aqueous solubility, blood brain barrier, plasma pro-tein binding, CYP2D6 binding and hepatotoxicity, were evaluated for these mol-ecules within human.

Results

The docking of known inhibitors and selected squalene synthase inhibitors into active site of S. aureus dehydrosqualene synthase enzyme was carried out using Scigress Explorer Ultra 7.7 software and Discovery studio 2.0, accelrys for com-parative analysis of binding residues (32, 33). The final docking score was calcu-lated in terms of Kcal/mole for each docking experiment. The analysis of docking score and Hydrogen bond interactions was done for all ligand molecules used in

Figure 1: 2D Molecular structures of the ligands/inhibitors.

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this study. (The molecules with their docking score and hydrogen bond interactions of these ligands with amino acid residues in active site of target proteins are given in supplementary data in Table I-IV). The potential energy of the dehydrosqualene synthase of S. aureus before docking was calculated by DS 2.0. (Data is available in supplementary Table III).

The docking analysis of standard drugs by Scigress Explorer Ultra 7.7 software and DS 2.0 is given in supplementary Table I and III, respectively. Molecular docking analysis of standard drug, B652 (Bisphosphonate derivative) showed the interaction by H-bond formation with Arg45, Tyr129 and Gln165 residues of dehydrosqualene synthase of S. aureus (Figure 2).

(A) Docking of Lapaquistat Acetate into Dehydrosqualene Synthase (CrtM) of S. aureus

Molecular interactions between lapaquistat acetate and dehydrosqualene synthase (CrtM), calculated the docking score. Different conformation states of this enzyme deposited in PDB (protein data bank) was taken in the molecular docking analysis with this test compound.It was found that the lapaquistat acetate made hydrogen bond with the residue His18 of crystal structure of this enzyme, PDB ID: 2ZCO during docking with docking score of 143.260 Kcal/mol. It also made hydrogen bond with the residue Asp114 of PDB ID: 2ZCP with docking score of 174.822 Kcal/mol (Figure 3a), with the residue Arg45 of PDB ID: 2ZCQ with the docking score of 100.337 Kcal/mol and with the residue His18 and Tyr129 of PDB ID: 2ZCR with the docking score of 155.874 Kcal/mol.

All the five different conformation states of dehydrosqualene synthase enzyme scored the docking energy between 175.224 to 100.337 Kcal/mol (Supplemen-tary data Table II). Comparative docking analysis by DS 2.0 has shown the involve-ment of Tyr 41, Tyr 129, Asn 168 and Arg 265 residues of PDB ID: 2ZCS with Lapaquistat acetate (Pubchem ID: 9874248) (Figure 3b).

(B) Docking of Squalestatin Analogs with Dehydrosqualene Synthase (CrtM) of S. aureus

(i) Squalestatin analog 2. Docking study has shown the hydrogen bonding of squalestatin analog 2 with His18, Arg45, Asp48, Asp52, Tyr129, Asn168, Asp172 and Asp176 residues of different conformation states of protein crystal struc-tures with a docking score values ranging from 208.730 Kcal/mol to 114.395

Figure 2: Docking of B652 (Bisphosphonate derivative, known inhibitor of dehydrosqualene synthase) to the active site of S. aureus Dehydrosqualene synthase (CrtM), PDB ID: 2ZCO

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Kcal/mol (Supplementary data Table II).Squalestatin analog 2 docked with dehy-drosqualene synthase (PDB ID: 2ZCQ) has shown the hydrogen bonding with three residues, Asp52, Tyr 129 and Asp 172 with a docking score of 208.730 Kcal/mol (Figure 4a). Comparative docking analysis by DS 2.0 has shown the involvement of His18, Arg 45, Tyr 129, Gln 161 and Asn 168 residues of PDB ID: 2ZCO with Squalestatin analog 2 (Figure 4b).

(ii) Squalestatin analog 15. Docking study with squalestatin analog 15 has shown hydrogen bonding with His18, Asp48, Asp52, Gln165, Asn168, Asp172, Glu175, Asp176, Tyr183 and Tyr248 residues of different conformation states of protein crystal structures with a docking score values ranging from 206.843 to 166.655 Kcal/mol (Supplementary data Table II). Squalestatin analog 15 docked with dehy-drosqualene synthase (PDB ID: 2ZCR) has shown the hydrogen bonding with four residues, Asp48, Tyr129, Asp172 and Asp176 with a docking score of 206.843 Kcal/mol. Comparative docking analysis by DS 2.0 has shown the involvement of Arg45, Tyr129, Asn168, Asp176 and Tyr183 residues of PDB ID:2ZCS with Squalestatin analog 15.

(iii) Squalestatin analog 20. Docking study with squalestatin analog 20 has shown hydrogen bonding with His18, Asp48, Gln165, Asn168, Arg265, Tyr129, Asp172, Asp176, Glu175 and Tyr183 residues of different conformation states of protein crystal structures with a docking score values ranging from 185.395 to 116.198 Kcal/mol (Supplementary data Table II). Squalestatin analog 20 docked with dehy-drosqualene synthase (PDB ID: 2ZCQ) has shown the hydrogen bonding with three residues, Asp 48, Asn168 and Asp176 with a docking score of 176.527 Kcal/mol. Comparative docking analysis by DS 2.0 has shown the involvement of His18, Phe22, Arg45 and Tyr 129 residues of PDB ID: 2ZCS with Squalestatin analog 20.

(iv) Squalestatin analog 25. Docking study with squalestatin analog 25 has shown hydrogen bonding with Gln165, Arg171, Asp172, Glu175, Asp176 and Arg265 residues of different conformation states of protein crystal structures with a dock-ing score values ranging from 197.088 to 170.609 Kcal/mol (Supple mentary data Table II). Squalestatin analog 25 docked with dehydrosqualene synthase (PDB ID: 2ZCP) has shown the hydrogen bonding with one residue, Asp172 with a docking score of 197.088 Kcal/mol. Comparative docking analysis by DS 2.0 has shown the involvement of His18, Tyr 41, Arg45, Val 133, Val137, Gln165, Asn168, Asp172, Glu175 and Arg265 residues of PDB ID: 2ZCO with Squalestatin analog 25.

Figure 3: (a) Lapaquistat acetate (Pubchem ID: 9874248) docked with dehydrosqualene synthase, PDB ID: 2ZCP. (b). Docking of Lapaquistat acetate (Pubchem ID: 9874248) to the active site of S. aureus (CrtM) Dehydrosqualene synthase, PDB ID: 2ZCS.

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(v) Squalestatin analog 32. Docking study with squalestatin analog 32 has shown hydrogen bonding with Asp48, Asp52, Gln165, Asp172 and Arg265 residues of different conformation states of protein crystal structures with a docking score val-ues ranging from 203.621 to 149.629 Kcal/mol (Supplementary data Table II). Squalestatin analog 32 docked with dehydrosqualene synthase (PDB ID: 2ZCP) has shown the hydrogen bonding with Asp 172 with a docking score of 203.621 Kcal/mol. Comparative docking analysis by DS 2.0 has shown the involvement of Phe22,

Arg45, Asp48, Tyr129, Val137, Gln165, Asn168, and Tyr183 residues of PDB ID: 2ZCO with Squalestatin analog 32.

(vi) Squalestatin analog 36. Docking study with squalestatin analog 36 has shown hydrogen bonding with Asp52, Asp172, Glu175 and Arg 265 residues of different conformation states of protein crystal structures with a docking score values ranging from 215.331 to 117.878 Kcal/mol (Supplementary data Table II). Squalestatin analog 36 docked with dehydrosqualene synthase (PDB ID: 2ZCP) has shown the hydrogen bonding with two residues, Asp 52 and Asp 172 with a docking score of 193.547 Kcal/mol. Comparative docking analysis by DS 2.0 has shown the involvement of His18, Asp48, Val137, Gln165, Asn168, and Arg171 residues of PDB ID: 2ZCS with Squalestatin analog 36.

Based on the comparative molecular docking study by Scigress Explorer Ultra 7.7 and Discovery Studio 2.0, the relative occurrence of His18, Arg45, Asp48, Asp52, Tyr129, Gln165, Asn168 and Asp172 residues of dehydrosqualene synthase (CrtM) of S.aureus was found to be high in H-bond formation with the inhibitors studied (Supplemen-tary data Table I-IV).

Bisphosphonate derivatives and farnesyl analogs used in the standard data set gave the docking score values ranging from 147.858 to 97.645 Kcal/mol. Where as the test compounds, lapaquistat acetate and squalestatin analogs used in the docking simulation into dehy-drosqualene synthase (CrtM) of S. aureus gave the docking score val-ues ranging from 215.331 to 100.337 Kcal/mol (Figure 5).

In Silico ADMET Analysis

In silico ADMET properties such as ADMET BBB level, absorp-tion, aqueous solubility, hepatotoxicity, CYP2D6, AlogP98 and PSA

Figure 4: (a) Squalestatin analog 2 (Pubchem ID: 460617) docked with dehydrosqualene synthase (PDB ID: 2ZCQ). (b) Docking of Squalestatin analog 2 (Pubchem ID: 460617) to the active site of S. aureus (CrtM) Dehydrosqualene synthase (PDB ID: 2ZCO).

Figure 5: Docking score of SQS inhibitors with dehydrosqualene synthase (CrtM) of Staphylococcus aureus.

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were studied for the standard compounds from standard data set and test com-pounds from test data set. An ADMET model was generated that predicts the human intestinal absorption (HIA) after oral administration of the inhibitors tested. The intestinal absorption model includes 95% and 99% confidence ellipses in the ADMET_PSA_2D and ADMET_AlogP98 plane (Figure 6). There are four predic-tion levels for the absorption of compounds as good (0), moderate (1), poor (2) and very poor (3). These levels are defined by the 95% (red line) and 99% (green line) confidence ellipsoids (Figure 6). The upper limit of PSA_2D value for the 95% con-fidence ellipsoid is at 131.62, while the upper limit of PSA_2D value for the 99% confidence ellipsoid is at 148.12 (Figure 6). Based on the in silico ADMET analysis it was found that the test compound (Pubchem ID: 9874248) fulfilled the ADMET descriptors criteria at the optimal level among the test compounds tested whereas all the compounds from the standard dataset fulfilled the ADMET descriptors criteria (Supplementary data Table V).

Discussion

Molecular docking study was done to find the binding site of SQS inhibitors within the dehydrosqualene synthase of S. aureus to inhibit its function. We have used lapaquistat acetate and squalestatin analogs reported as SQS inhibitors in human and animal models but not studied in context to dehydrosqualene syn-thase (CrtM) of S. aureus to study the molecular interaction between them. From the frequency of residue occurrence in the formation of hydrogen bonding with inhibitors studied, it was found that the His18, Arg45, Asp48, Asp52, Tyr129, Gln165, Asn168 and Asp172 interacted with comparatively high frequency. Comparative molecular docking analysis by DS 2.0 has also shown the similarity with the results obtained by Scigress Explorer Ultra 7.7.It was observed that the bisphosphonates derivatives and farnesyl analog has shown the interaction with His18, Arg45, Gln165 and Asp172 residues of dehydrosqualene synthase (CrtM) of S. aureus via hydrogen bonding in the standard dataset. Crystal structures of S. aureus dehydrosqualene synthase (CrtM) bound with bisphosphonates has also shown the involvement of aspartic acid(D) and asparagine(N) residues in hydro-gen bonding with these inhibitors. Docking study has shown the interaction of acidic and polar residues of dehydrosqualene synthase enzyme with lapaquistat acetate and squalestatin analogs.

Figure 6: Plot of Polar Surface Area (PSA) vs.LogP for a standard and test set showing the 95% and 99% confidence limit ellipses corre-sponding to the Blood Brain Barrier and Intes-tinal Absorption models.

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The results has shown a good docking score ranging from 215.331 to 100.337 Kcal/mol with test compounds, lapaquistat acetate and squalestatin analogs (Figure 5). Standard compounds, bisphosphonates and farnesyl analogs gave the docking score ranging from 147.858 to 97.645 Kcal/mol during molecu-lar docking (Figure 5). It was also found that all the crystal structures of dehy-drosqualene synthase had variations in the docking score and the binding residues due to differences in the molecular conformations of this enzyme. The In silico ADMET analysis predicted that the test compound, Lapaquistat (Pubchem ID: 9874248) fulfilled the ADMET descriptors criteria with the compounds taken in the study (Supplementary data Table V).

Conclusions

The present study implicated that the residues involved in interaction with the test compounds used has similarity with the standard compounds at the binding pocket of dehydrosqualene synthase enzyme. These residues can be considered to being involved in functionality of this enzyme and can be used as a target site for the development of dehydrosqualene synthase (CrtM) inhibitors. It is to be mentioned that all the contributing amino acids in interaction possess the same set of common physico-chemical properties that imparts stability to the interaction. These amino acids are hydrophilic, polar, charged and turn like structures. The bonding takes place at the polar surface of this enzyme. This further confirms the functionality of the residues which makes the active site of this enzyme. Binding of the inhibitors used in the study at the active site of dehydrosqualene synthase (CrtM) confirms this aspect. In silico ADMET analysis was done to predict the ADMET properties of the test compounds and compared with the standard drugs.

These findings can be exploited to design dehydrosqualene synthase specific inhib-itors. Further studies with these test compounds in vitro and in vivo with respect to inhibition of dehydrosqualene synthase (CrtM) of S. aureus could give more insight into the action of these inhibitors. Hence, this would help in the develop-ment of new drugs that could target this virulence factor of S. aureus.

Supplementary Material

Supplementary material dealing with the docking results by Scigress Explorer Ultra 7.7 and Discovery Studio 2.0 is provided in Table I-IV. In silico ADMET analysis results for the test and standard ligands is also available in the supplementary data in Table V at no charge from the authors directly; the supplementary data can also be purchased from Adenine Press for US $50.00.

Acknowledgements

Financial support of Department of Biotechnology [DBT] under BTISnet program, Govt. of India is gratefully acknowledged. The authors are also grateful to Ms. Akansha Saxena, Scientist, ICMR, New Delhi for help in DS 2.0 analysis.

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Date Received: December 16, 2009

Communicated by the Editor Ramaswamy H. Sarma