Research Article

Received: 20 April 2013, Accepted: 29 April 2013

ISSN: 2321-2969

Int. J. Pharm. Biosci. Technol.

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International Journal of Pharma Bioscience and Technology; Volume 1, Issue 1, May 2013, Pg 34-49

Journal home page: www.ijpbst.com

DEVELOPMENT OF PHARMACOPHORE MODEL FOR S. AUREUS DNA GYRASE INHIBITORS

Arundhati C. Lele, Nilesh R. Tawari, Mariam S. Degani*
Pharmaceutical Chemistry Research Laboratory, Department of Pharmaceutical Sciences and Technology, Institute of Chemical Technology, N. P. Marg, Matunga, Mumbai 400019, India Corresponding Author* E-mail address- msdegani@gmail.com

ABSTRACT: Pharmacophore mapping studies were undertaken for a series of molecules belonging to indazole, pyrazole and their congeners as inhibitors of S. Aureus DNA gyrase. A four point pharmacophore with two aromatic rings (R), one lipophilic/hydrophobic group (H) and one positive ionic feature (P) as pharmacophoric features was developed. The pharmacophore hypothesis yielded a statistically significant 3D-QSAR model, with correlation coefficient of r2 = 0.681 for training set molecules. The model so generated showed good predictive power with correlation coefficient for an external test set of fifteen molecules. The geometry and features of the pharmacophore will be useful for ligand based design of potential DNA gyrase inhibitors. Key words: Pharmacophore, 3D-QSAR, DNA gyrase, S. Aureus. INTRODUCTION Antimicrobial resistance is a growing threat worldwide. Staphylococcus aureus infections are a major cause of morbidity and mortality in community and hospital settings.[1] The emergence of methicillin resistant and vancomycin resistant strains of S. Aureus represents an enormous threat to public health with MRSA accounting for as many as 50% of infections.[2] S. Aureus strains that combine resistant and virulent genes have become a major treatment problem in Europe and the U.S.[3] Hence there is an ever increasing need for the development of newer antibiotics to treat S. aureus infections. DNA gyrase is a prokaryotic specific type topoisomerase and has attracted considerable attention from the pharmaceutical industry ever since the discovery of Nalidixic acid (DNA gyrase inhibitor) in the early 1960s. Other well-known DNA gyrase inhibitors include quinolones, coumarins and cyclothialidines.[4] Bacterial DNA gyrase is involved in the vital processes of DNA replication, transcription, and recombination and catalyzes the ATP-dependent introduction of Lele et al negative supercoils into bacterial DNA as well as the decatenation and unknotting of DNA. [5] Ligand based drug design approaches like pharmacophore mapping and QSAR can be used in drug discovery in several ways; the rationalization of activity trend in molecules under study, to predict the activity for new compounds, for database search studies in search of new hits and to identify important features for activity. This paper describes the development of a robust ligand based 3D- pharmacophore hypothesis using Pharmacophore Alignment and Scoring Engine (PHASE) for S. aureus DNA gyrase. Ligands selected for the development of the pharmacophore model and the subsequent generation of atom based 3D-QSAR were indazoles and pyrrazoles analogues acting as DNA gyrase inhibitors and the alignment obtained from the generated pharmacophoric points is used to derive an atom based 3D-QSAR model. The pharmacophore thus developed imparts information about important features for DNA gyrase inhibitors and the geometry has the ability to mine 3D virtual databases of drug like Pg. 34

Int. J. Pharm. Biosci. Technol. molecules. Furthermore, the contours generated from QSAR studies highlight the structural features required for DNA gyrase inhibitory activity and are useful for further design of more potent inhibitors. MATERIALS AND METHODS Biological data A set of 63 compounds shown in Table 1(a) to Table 1(m), belonging to the family of indazole and pyrrazole analogues reported as DNA gyrase inhibitors [6-10] were used for the pharmacophore generation. The negative logarithm of the measured IC50 value as pIC50 was used in the 3DQSAR study. The complete set was divided into a training set (48 compounds) and a test set (15 compounds) using randomization as well as chemical and biological diversity.

Table 1. Structures and the inhibitory concemtrations of the set of molecules utilized for generation of pharmacophore model. Table 1(a) [6] No Structure MIC (g/ml)

1.

64

2.

0.25

Novobiocin Table 1(b) General structure 1.[6]

No.

R1

MIC (g/ml)

3.

32

4.

16

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H3C 5. O

O NH

64

HN H3C 6. O HN O

64 NH

7.

32

8.

64

32

10.

32

11.

16

12. H3C 13. O O

16

H N

Table 1(c) General structure 2 [6]

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Int. J. Pharm. Biosci. Technol.

Structure No.

R1

MIC (g/ml)

14.

64

15.

128

16.

32

17.

Table 1(d) [7] Structure No. Structure MIC (g/ml)

18.

19.

20.

21.

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Int. J. Pharm. Biosci. Technol. Table 1(e) General structure 3 [7]

Structure No.

R3

MIC (g/ml)

22. (E Stereochemistry) 23. (Z Stereochemistry)

24.

(E Stereochemistry)

25. (Z Stereochemistry)

Table 1(f) General structure 4 [8]

Structure No.

MIC (g/ml)

26.

27.

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Int. J. Pharm. Biosci. Technol.

28.

29.

30.

32

31.

16

32.

33.

34.

0.125

Sparfloxacin Table 1(g) General Structure 5 [8]

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Structure No.

MIC (g/ml)

35.

36.

128

37.

32

38.

Table 1(h) General Structure 6 [9]

Structure No.

R1

R2

MIC (g/ml)

39.

64

40.

64

48.

CH3

32

42.

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43.

44.

45.

46.

Table 1(i) General Structure 7 [9]

Structure No.

R1

R2

MIC (g/ml)

47.

CH3

64

48.

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Int. J. Pharm. Biosci. Technol. Table 1(j) [10] Structure No. Structure MIC (g/ml)

49.

64

Table 1(k) General Structure 8 [10]

Structure No.

R1

H-R2

MIC (g/ml)

50.

51.

52.

16

53.

54.

55.

64

56

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Int. J. Pharm. Biosci. Technol. Table 1(L) General Structure 9 [10]

Structure No.

R1

H-R2

MIC (g/ml)

57.

58.

59.

Table 1(m) General Structure 10 [10]

Structure No.

R1

H-R2

MIC (g/ml)

60.

3- Cl

61.

3- Cl

62.

NH

3- Cl

63.

NH

3- Cl

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Int. J. Pharm. Biosci. Technol. Generation of Common Pharmacophore Hypothesis (CPH) The pharmacophore hypothesis and alignment based on it was carried out using PHASE, version 2.0, Schrdinger, LLC, New York, NY, 2005 installed on AMD Athelon workstation.[11] The structures were imported from the project table in the Develop Pharmacophore Hypothesis panel and geometrically refined (cleaned) using Ligprep. Conformations were generated by MCMM/LMOD method using maximum of 2000 steps with distance dependent dielectric solvent model and OPLS-2005 force field. All the conformers were subsequently minimized using truncated Newton conjugate gradient minimization up to 500 iterations. For each molecule a set of conformers with maximum energy difference of 30 kcal/mol relative to the global energy minimum were retained. A redundancy check of 2 in the heavy atom positions was applied to remove duplicate conformers. Pharmacophore features; hydrogen bond acceptor (A), hydrogen bond donor (D), hydrophobic group (H), negatively charged group (N), positively charged group (P), aromatic ring (R), were defined by a set of 4 chemical structure patterns as SMARTS queries and assigned one of three possible geometries, which define physical characteristics of the site: Pointthe site is located on a single atom in the SMARTS query. Vectorthe site is located on a single atom in the SMARTS query, and assigned directionality according to one or more vectors originating from the atom. Groupthe site is located at the centroid of a group of atoms in the SMARTS query. For aromatic rings, the site is assigned directionality, defined by a vector that is normal to the plane of the ring. Active and inactive thresholds were applied to the dataset (Table 1.) to yield 11 actives and 12 inactives which were used for the pharmacophore generation and subsequent scoring. Common pharmacophoric features were then identified from a set of variants, a set of feature types that define a possible pharmacophore, using a tree based partitioning algorithm with maximum tree depth of 4. The final size of the pharmacophore box was 1 to optimize the number of final common pharmacophore hypotheses (CPH). These CPHs were examined using a scoring function to yield the best alignment of the active ligands using overall maximum RMSD value of 1.2 with default options for distance tolerance. The quality of alignment was measured by a survival score, which was defined using following equation. [12] Lele et al S= WsiteSsite + WvecSvec + WvolSvol + WselSsel + Wrewm Where Ws are weights and Ss are scores Ssite represents alignment score, the root mean square deviation in the site point position. Svec represents vector score, and averages cosine of the angles formed by corresponding pairs of vector features in the aligned structures. Svol represents volume score based on overlap of Van der Waals models of non hydrogen atoms in each pair of structures. Ssel represents selectivity score, and accounts for what fractions of molecules are likely to match the hypothesis regardless of their activity towards receptor. Wsite, Wvec, Wvol, Wrew have default value of 1.0 while Wsel has default value of 0.0. In the hypothesis generation default values have been used. Wrewm represents reward weights defined by m-1 where m is the number of actives that match the hypothesis. The assessment of the generated CPHs was performed by correlating the observed and estimated activity for the internal set of 48 training molecules and external set of 15 test molecules using partial least square analysis. The partial least square (PLS) regression was carried out using PHASE with maximum of 4 PLS factors using pharmacophore based model, with grid spacing of 1. The CPH with best predictive value and significant statistical data was chosen for alignment of molecules and used for further 3DQSAR studies. QSAR Model Building A training set of 48 molecules were selected randomly, incorporating biological and chemical diversity, and were used to generate atom-based QSAR models for all hypotheses using a grid spacing of 1.0 . Models containing four or more PLS factors tended to fit the pIC50 values beyond their experimental uncertainty and thus, only one, two and three factor models were considered. Each of these models were validated using an external test set of fifteen molecules which were not considered during model generation. RESULTS AND DISCUSSION Pharmacophore models containing three and four sites were generated using a terminal box size of 1 with twelve highly active molecules selected [610] belonging to indazole, pyrazole and their congeners, using a tree based partition algorithm. The three featured CPHs were rejected, as they Pg. 44

Int. J. Pharm. Biosci. Technol. were unable to define the complete binding space of the selected molecules. A total of 10,487 probable four featured CPHs belonging to seven types HHPR, AHPR, HHRR, AHHR, AHRR, AHHP and HPRR were subjected to stringent scoring function analysis, with respect to actives using default parameters for site, vector, and volume. Hypotheses emerging from this process were subsequently scored with respect to the seven inactives, using a weight of 1.0. The hypotheses which survived the scoring process were used to build an atom-based QSAR model. The summary of statistical data of the best CPHs labeled CPH1 to CPH6 with their survival scores is listed in Table 2.

Table 2. Summary of QSAR results for six best CPHs with survival scores
CPH1 (HPRR) Survival score SD r2 F P RMSE Q2 Pearson-R 3.136 0.422 0.681 31.3 5.413e-11 0.452 0.525 0.741 CPH2 (HHPR) 3.136 0.444 0.647 26.9 4.949e-10 0.466 0.510 0.720 CPH3 (HHRR) 3.092 0.372 0.729 35.1 3.686e-11 0.486 0.449 0.754 CPH4 (AHHR) 3.026 0.443 0.648 27.1 4.429e-10 0.471 0.4842 0.736 CPH5 (AHRR) 2.872 0.386 0.699 33.4 2.644e-11 0.501 0.414 0.646

SD = Standard deviation of the regression, r2 = Value of r2 for the regression, F = Variance ratio P = Significance level of variance ratio, RMSE = Root-mean-square error, Q2 = Value of Q2 for the predicted activities, Pearson-R = Correlation between the predicted and observed activity for the test set. Consistent external predictivity was observed for CPH1 for each combination as compared to others. The CPH1 showed a statistically significant r2 value for training set (0.681), excellent predictive power with Q2 of 0.525 and a Pearson-R value of 0.741. Hence, the hypothesis CPH1 with two aromatic ring (R), one lipophilic/hydrophobic group (H) and one positive ionic feature (P) as pharmacophoric features was retained for further studies. Actual and predicted values of the training set and test set molecules are given in Table 3. Figure 1 shows the alignment of the molecules under study along with CPH1 while Figure 2 shows the gemotry of the pharmacophore generated. The distances and angles between the pharmacophoric features are depicted in Figure 3(a) and 3(b) respectively. Figures 4(a) and Figure 4(b) shows the graphs of actual v/s. predicted activity for training and test set molecules.

Table 3. Actual and predicted values of the dataset Structure No. 1 2 3 4 5 6 7 8 9 Lele et al Biological activity -2.283 0.389 -1.995 -1.626 -2.166 -2.166 -1.894 -2.195 -1.866 Predicted activity -2.34 0.31 -1.80 -1.76 -1.56 -1.92 -1.72 -1.83 -1.72 Structure No. 33 34 35 36 37 38 39 40 41 Biological activity -0.359 0.497 -0.669 -2.599 -1.994 -1.094 -2.302 -2.363 -1.964 Predicted activity -0.87 0.98 -1.28 -1.49 -1.53 -1.51 -2.34 -2.29 -2.25 Pg. 45

Int. J. Pharm. Biosci. Technol. 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 -1.866 -1.551 -1.577 -0.949 -2.211 -2.474 -1.874 -0.937 -0.949 -0.937 -0.359 -0.345 -0.991 -0.991 -1.273 -0.972 -0.57 -1.267 -0.646 -0.66 -1.867 -1.561 -0.966 -1.56 -1.50 -1.56 -1.42 -1.80 -1.41 -1.84 -1.22 -1.57 -1.38 -0.87 -0.90 -1.41 -1.10 -0.99 -1.47 -0.93 -0.87 -0.90 -0.87 -0.97 -0.97 -1.07 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 -1.013 -0.969 -0.99 -0.966 -0.359 -2.265 -1.267 -2.283 -0.969 -0.668 -1.571 -0.68 -1.011 -2.138 -0.654 -0.969 -1.312 -0.955 -0.968 -0.653 -0.668 -0.654 -1.11 -1.11 -1.04 -0.96 -0.99 -2.06 -0.97 -2.34 -0.93 -0.93 -1.25 -0.73 -1.30 -1.25 -0.86 -1.12 -0.98 -1.13 -1.14 -1.21 -0.98 -1.35

Fig. 1. CPH1 based alignment DNA gyrase inhibitors.

Fig. 2. Geometry of the pharmacophore- P9 is the positive ionic feature represented as blue sphere; R11 and R12 are the aromatic ring features represented in orange torus and H4 is the hydrophobic feature represented in green sphere. Pg. 46

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Fig. 3(a) CPH1 features and distances. Fig. 4(b). Scatter plots for the predicted and experimental pIC50 values for the DNA gyrase QSAR model applied to the test set. The pharmacophore contains two aromatic ring (R) features mapping on the pyrazole heterocycle and benzene ring, one positive ionic feature mapping on one of the nitrogens in the piperazine ring and one lipophilic/ hydrophobic feature mapping on the para-chloro atom in the distal part of molecule. When the QSAR model was visualized in the context of one of the most active molecule (Structure 21) blue cubes, favorable for the activity, were observed in context of the pharmacophoric features thus corroborating the findings of pharmacophore as observed in Fig. 5. Fig. 3(b) CPH1 features and angles.

Fig. 5. QSAR model visualization for DNA gyrase B of S. aureus. CONCLUSION The current work involves development of predictive pharmacophore hypothesis (CPH) for S. aureus DNA gyrase inhibitors using PHASE and performing the QSAR analysis of the model. Best CPH was selected primarily on the basis of its internal and external predictive capabilities. The Pg. 47

Fig. 4(a). Scatter plots for the predicted and experimental pIC50 values for the DNA gyrase QSAR model applied to the training set.

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Int. J. Pharm. Biosci. Technol. generated CPH consists of two aromatic rings, one lipophilic/hydrophobic group and one positive ionic feature. The pharmacophore hypothesis yielded statistically significant 3D-QSAR model. The developed pharmacophore model will aid in the rational design of DNA gyrase inhibitors. ACKNOWLEDGEMENTS Author A. C. Lele is thankful to Council of Scientific and Industrial Research, Delhi, India and N. R. Tawari is thankful to Department of Biotechnology, Delhi, India for financial assistance.

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Int. J. Pharm. Biosci. Technol. ___________________________________________ How to cite this article APA style Lele, A. C., Tawari, N. R., & Degani, M. S. (2013). Development of pharmacophore model for S. Aureus DNA-gyrase inhibitors. International Journal of Pharma Bioscience and Technology, 1(1), 3449. Elsevier Harvard style Lele, A.C., Tawari, N.R., Degani, M.S., 2013. Development of pharmacophore model for S. Aureus DNA-gyrase inhibitors. Int. J. Pharm. Biosci. Technol. 1, 3449. Vancouver Style Lele AC, Tawari NR, Degani MS. Development of pharmacophore model for S. Aureus DNAgyrase inhibitors. Int. J. Pharm. Biosci. Technol. 2013;1(1):3449. To receive bibliographic information in RIS format (For Reference Manager, ProCite, EndNote): Send request to: ijpbst@yahoo.com ___________________________________________

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