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Cite this article: Khera MK, Mathur T, Barman TK, Ramkumar G, Kumar M, et al. (2017) Antibacterial Activity of a Novel 1,2,4-Triazolo [4,3-A] Pyrimidine Oxazolidinone against Broad Spectrum of Gram Positive Pathogens and Molecular Modeling Studies for its Interaction with Ribosome. JSM Microbiology 5(1): 1034.

*Corresponding authorManoj Kumar Khera, Department of Medicinal Chemistry, Daiichi Sankyo India Pharma Private Limited, Village Sarhaul, Sector – 18, Gurgaon – 122 015, India, Tel: 91-9818337315; Fax: 91-124-2397546; Email:

Submitted: 01 March 2017

Accepted: 06 April 2017

Published: 08 April 2017

Copyright© 2017 Khera et al.

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Keywords•Oxazolidinone•Antibacterial•MRSA

Review Article

Antibacterial Activity of a Novel 1,2,4-Triazolo [4,3-A] Pyrimidine Oxazolidinone against Broad Spectrum of Gram Positive Pathogens and Molecular Modeling Studies for its Interaction with RibosomeManoj Kumar Khera1*, Tarun Mathur2, Tarani Kanta Barman2, Ramkumar G2, Manoj Kumar2, Dilip J. Upadhyay2, Tarun Jain1, Om Prakash3, Ian A. Cliffe1, Smita Dube2, and V. Samuel Raj2

1Department of Medicinal Chemistry, Daiichi Sankyo India Pharma Private Limited, India2Department of Microbiology, Daiichi Sankyo India Pharma Private Limited, India3Department of Chemistry, Kurushetra University, India

Abstract

Oxazolidinones are known to inhibit bacterial protein biosynthesis and act against a wide spectrum of Gram-positive bacteria. Herein, we report a novel investigational oxazolidinone compound 1, which showed potent in vitro activity against Staphylococcus aureus, Staphylococcus epidermidis, Vancomycin resistant enterococci (VRE), Streptococcus pneumoniae, Streptococcus pyogenes and other pathogens. The MIC of compound 1 was 0.125 µg/ml against S.aureus ATCC 25923, whereas that of linezolid was 1 µg/ml. Compound 1 showed faster inhibition of bacterial protein synthesis than Linezolid in S.aureus ATCC 25923. Molecular modeling studies indicated that compound 1 exhibited binding to the ribosome in a similar manner to that of Linezolid and Ranbezolid but with additional interactions from its triazolopyrimidine and thiophene rings which are probably responsible for its stronger in vitro activity.

INTRODUCTIONBacterial infection due to Gram positive bacteria such as

methicillin resistant Staphylococcus aureus (MRSA), methicillin-resistant Staphylococcus epidermidis (MRSE) and vancomycin resistant enterococci (VRE) have emerged as a major public health problem across the globe. The emergence of antimicrobial resistance among these pathogens has become troublesome for patients and clinicians. In the year 2014, the agency for healthcare research & quality reported 23,000 deaths due to MRSA in the United States alone. Staphylococci are an important cause of infection of the bloodstream, cardiac valves, implanted devices and skin and have repercussions on mortality and morbidity. Streptococcus pathogens are a major cause of mortality and morbidity associated with bacterial pneumonia, bacteremia,

sinusitis, skin infections and bacterial meningitis. The problem is more severe in the hospital setting, where MRSA has emerged. MRSA was first reported in 1961 and has since become a major nosocomial pathogen worldwide. The treatment of staphylococcal infections is becoming difficult due to the increasing emergence of resistance to beta-lactams and other antimicrobials, including reduced susceptibility to glycopeptides [1-6]. In recent years, there has been an increase in infections caused by multiple pathogens and the situation has become quite alarming with the recent emergence of vancomycin-intermediate S.aureus (VISA), vancomycin resistant enterococci (VRE), etc.

Oxazolidinones are the only new class of synthetic antibiotics to be developed in the past 30 years and they act against a wide spectrum of primarily Gram-positive bacteria. Oxazolidinones

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have a mechanism of action which involves inhibiting the initiation of protein synthesis at a site different from other known protein synthesis inhibitors. These have activity against many antibiotic resistant pathogens, including those cross resistant to protein synthesis inhibitors [7]. Early studies on the mechanism of action of oxazolidinones have shown no effect on DNA and RNA synthesis [8-10].

Linezolid was the first antibacterial drug in the oxazolidinone class for the treatment of a number of infections including complicated skin and skin structure infections caused by S. aureus (MRSA and MSSA), S. pyogenes or S. agalactiae; uncomplicated skin and soft tissue infections caused by S. aureus or S.pyogenes; hospital acquired pneumonia caused by S. aureus; and community acquired pneumonia caused by S.pneumoniae, S. aureus or vancomycin-resistant Enterococcus faecium [11]. Subsequently, Tedizolid phosphate was developed for the treatment of acute bacterial skin and skin structure infections; however, it cannot be used for neutropenic patients [12]. Over a period of time, many investigational oxazolidinones have been reported which have shown a good spectrum of activity against Gram-positive pathogens [7,13-16].

The crystal structure of Linezolid bound to the 50S ribosomal unit was reported by Duffy et al., from Rib-X pharmaceuticals [17]. Along with the identification of key interactions, a significant observation was the morpholine ring of Linezolid appearing not to make significant interactions with the ribosome and its replacement with a wide variety of other groups was found to result in an improvement in antibacterial activity.

In this paper we report the in vitro properties against Staphylococci and Streptococci and mode of action of the investigational oxazolidinone compound 1 (Figure 1) wherein morpholine ring of Linezolid has been replaced with a 3-(thiophen-2-yl)[1,2,4]triazolo[4,3-a]pyrimidine moiety. In addition, we report the molecular docking studies used to explain the interaction of compound 1 with the ribosome.

MATERIALS AND METHODS

Bacterial strains and antibiotics

The set of Gram-positive facultative and fastidious ATCC strains used in this study are shown in (Table 1,2). The S.aureus ATCC 25923 strain was used for kill kinetics and macromolecule synthesis inhibition studies. The oxazolidinone compound 1 was synthesized in-house [18] and other antibiotics were procured from commercial sources. The radiolabeled compounds 3H-thymidine, 3H-uridine, 14C-isoleucine, 14C-acetate, and 3H-N-

acetylglucosamine were procured either from Perkin Elmer (USA) or the Board of Radiation and Isotope Technology (India).

Minimum inhibitory concentrations (MICs)

The MICs for Linezolid and Compound 1 were performed against Gram-positive facultative and fastidious strains according to Clinical and Laboratory Standard Institute (CLSI) guidelines (CLSI, 2012).

Time-kill kinetic studies

The time-kill kinetic studies were performed as per Hoellman et al. [19]. The S. aureus ATCC 25923 was exposed to compound 1 or Linezolid at concentrations ranging from 0.5µg/ml to 16 µg/ml. Time–kill kinetic data were determined from the reduction in viable count (Log10cfu/ml) at 2h, 4h and 8h and compared with 0 h. Antibiotics were considered bactericidal at the lowest concentration that reduced the original inoculum by >3 log10 cfu/ml (99.9% killing) and bacteriostatic if <3 log10cfu/ml.

Macromolecular synthesis inhibition studies in S. aureus ATCC 25923

The macromolecular biosynthesis inhibition in S. aureus ATCC 25923 was studied as described by Oliva et al. [20], with some modifications by Kalia et al [10]. The S.aureus ATCC 25923 was grown in Muller Hinton Broth (MHB) medium. Radioactive precursors (1 μCi/ml for 3H-labeled and 0.1 μCi/ml for 14C-labeled compounds) were added during the early logarithmic phase (OD600 0.3) and after 5 minutes inhibitors were added at their minimum inhibitory concentration (MIC), as determined by a microdilution method. The macromolecules (DNA, RNA, protein, fatty acid and cell wall) were precipitated with trichloroacetic acid (final 5%, wt/vol) and filtered on glass fibre filters (1.0 μM A/B glass multi-well filter plates, Pall Corporation). The plates were dried overnight at 37oC. The quantification of radioactivity was done using OptiPhase safe scintillation fluid and counting performed with a scintillation counter (Wallac Ltd).

Molecular modeling studies for interaction of compound 1 with ribosome

The molecular modeling studies were carried out using Maestro 9.3 molecular modeling software from Schrodinger (Schrodinger). The crystal structure of the E. coli 50S large subunit of ribosome (PDB ID 2AW4) [21] was used to build a working model based on residues within 30 Å of A2541 [22]. Hydrogen atoms and force field parameters were added to the crystal structure using the Protein Preparation Wizard in

Linezolid Compound 1

N NO

O

NH

OF

ON

N

NN

SN

OO

NH

CH3

OF

Figure 1 Structures of Linezolid and Compound 1.

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Table 1: In vitro susceptibility of compound 1 against facultative Gram positive pathogens.

MIC µg/mlS.NO ORGANISMS Linezolid Compound 1

1 S.aureus ATCC 25923 1 0.1252 S.aureus ATCC 13709 Smith 2 0.253 S.aureus ATCC 29213 4 0.54 MRSA ATCC 43300 2 0.255 MRSA 562 1 0.256 S.aureus2 (PVL+ve) 2 0.257 S.aureus 1199B (Nor pump+ve) 2 0.258 MRSA WCU H29 2 0.259 S.aureus ATCC Newman 25904 1 0.125

10 MRSA ATCC BAA 39 1 0.12511 S.aureus FDA209P 1 0.0612 S.aureus DB00026 1 0.12513 MRSA 252 BAA 1720 1 0.12514 S.epidermidis ATCC 12228 0.5 0.0315 MRSE ATCC 35983 1 0.12516 MRSE ATCC 35984 1 0.0617 S. epidermidis ATCC 14990 0.5 0.0618 E. faecalis ATCC 29212 2 0.2519 E.faecalis ATCC 51299(VRE&HLAR) 2 0.0620 E.faecalis ATCC 19433 2 0.2521 E.faecium ATCC 49224 1 0.12522 E.faecium 6A (VRE) 1 0.12523 E.faecium ATCC 19434 1 0.12524 E.faecium 06076VRE 1 0.12525 E.faecium ATCC 35667 1 0.12526 E.coli 7632 >8 >827 E.coli 120 (Acr-ve) 4 >8

Table 2: In vitro susceptibility of compound 1 against fastidious Gram positive pathogens.

MIC (μg/ml)

ORGANISMS Linezolid Compound 1

1 S. pyogenes ATCC 19615 1 0.125

2 S. pyogenes 203 C 1 0.125

3 S. pyogenes 3814 erm A 0.5 0.06

4 S. pyogenes 1721erm A 1 0.125

5 S. pyogenes 2534 erm B 1 0.125

6 S. pyogenes 2569 erm A 0.5 0.06

7 S. pyogenes 2368 erm B 0.5 0.06

8 S. pyogenes 2033 erm B 0.5 0.06

9 S. pyogenes 2534 NovRerm B 1 0.125

10 S. pyogenes ATCC 12344 1 0.125

11 S. salivarius ATCC 13419 1 0.125

12 S. viridans 1263 2 0.125

13 S. mitis ATCC 49456 1 0.06

14 S. mutans 956 4 0.25

15 S. salivaries 1061 1 0.125

16 S. sanguis SS982 1 0.06

17 S. viridans 659 1 0.125

18 S. agalactiae ATCC 13813 1 0.125

19 S. agalactiae ATCC 27956 1 0.125

20 S. pneumoniae ATCC 49619 1 0.125

21 S. pneumoniae 406081 erm B 0.5 0.06

22 S. pneumoniae R 6 0.5 0.06

23 S. pneumoniae 6303 1 0.125

24 S. pneumoniae 3579 erm 0.5 0.06

25 S. pneumoniae 994 mef 2 0.125

26 S. pneumoniae MA 80 erm 0.5 0.06

27 S. pneumoniae ATCC 49619 Nov R 1 0.125

Maestro. An active site model was generated using the Receptor Grid Generation protocol by selecting A2451 as the center of the grid. The compound 1 was docked into the generated receptor grid using Glide docking module [23] and the binding poses generated in this manner were analyzed.

RESULTSAntimicrobial activity of compound 1

The in vitro results of the testing of compound 1 and linezolid against a broad spectrum of pathogens causing skin infection, bacteremia and pneumonia are given in (Tables 1,2). The MICs of compound 1 and linezolid against S.aureus ATCC 25923, S.aureus ATCC 29213, MRSA ATCC 43300 and MRSA WCU H29 were 0.125, 0.125, 0.25 and 0.25 µg/ml and 1, 4, 2 and 2 µg/ml, respectively. The MICs of compound 1 and linezolid against S.pyogenes ATCC 19615, S.pyogenes ATCC 12344, S.pneumoniae ATCC 49619 and S.pneumoniae 6303 were 0.125, 0.125, 0.125 and 0.125 µg/ml and 1, 1, 1 and 1 µg/ml, respectively. The detailed MICs of compound 1 and linezolid against a broad spectrum of other strains are given in (Tables 1,2).

Kill kinetics of Compound 1 and Linezold against S. aureus

In time kill study, both compound 1 and linezolid showed bacteriostatic effects against S. aureus 25923 up to 8 h.

Macromolecular synthesis inhibition

Compound 1 was found to be a potent inhibitor of protein synthesis in S.aureus 25923 as it inhibited the incorporation of the specific radiolabeled precursor [14C] isoleucine into the nascent polypeptide chain during protein synthesis (Figure 2). The effect of compound 1 was faster than that of linezolid (30min vs. 60 min). DNA and RNA synthesis were not inhibited by compound 1 or linezolid in S. aureus 25923. However, a small amount of inhibition of cell wall and lipid synthesis was observed for both compounds at 60 minutes in a non-specific manner (Figure 2a and 2b). The macromolecular synthesis inhibition data confirmed compound 1 to be more potent protein synthesis inhibitor than Linezolid. In another experiment, Linezolid inhibited the protein synthesis of S. aureus with less potency, but stronger protein synthesis inhibition was observed with compound 1 (Figure 2c). Compound 1 inhibited protein synthesis (approximately 50% inhibition) at 0.25 μg/ml concentration, while 1 μg/ml of Linezolid was needed to show similar protein synthesis inhibition (Figure 2c).

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Molecular interaction of the compound 1 with ribosome

Since compound 1 specifically inhibited bacterial protein synthesis, it was important to understand the nature of binding mode of compound 1 to the bacterial ribosome. Compound 1 was docked in the crystal structure of the 50S ribosomal unit of E. coli (Figure 3) with the result that its binding mode was found to be similar to that of Linezolid and Ranbezolid [10]. The oxazolidinone ring forms a stacking interaction with the face of the uracil base of U2504. Other residues interacting with the oxazolidinone ring are the sugar of G2505 and the base of G2061. The amide side-chain of compound 1 is folded over the oxazolidinone ring and the amide N-H makes a hydrogen bond interaction with the 5’-oxygen atom of G2505. The phenyl ring proximal to the oxazolidinone ring is stabilized by the bases A2451 and C2452, while the thiophene ring is stabilized by Van der Waals interactions with the sugar residues A2451, C2452 and U2506. Additionally, the triazolopyrimidine ring is stabilized by residues A2451, U2584 and U2585.

DISCUSSION The oxazolidinones are inhibitors of bacterial protein

synthesis with a unique mechanism of action [7,24,25]. Compound

1 was confirmed to act by the same unique mechanism by use of a macromolecular synthesis inhibition assay. As expected from an oxazolidinone class of molecules, both compound 1 and linezolid showed in vitro activity against the Gram-positive pathogens tested in this study. Compound 1 showed at least a 4- to 8- fold more potent inhibition of staphylococci, enterococci and streptococcus strains than linezolid (Table 1 and 2). The improved in vitro activity of compound 1 may be attributed to additional interactions (shown by modeling studies) of its thiophene and triazolopyrimidine rings with the ribosome, which are absent in linezolid. The in vitro activity of compound 1 is comparable to an earlier compound Ranbezolid [10] from our laboratory.

Although both compound 1 and Linezolid showed bacteriostatic effect in kill kinetic studies, compound 1 showed a more rapid protein synthesis inhibition in S.aureus than Linezolid. The inhibition of protein synthesis by compound 1 and Linezolid are specific oxazolidione class effects, as they both bind to the 50s ribosome [14,22,26,27].

CONCLUSIONCompound 1 is a potent protein synthesis inhibitor with

in vitro activities against a broad spectrum of pathogens and, therefore, has the potential to be developed as an inhibitor of

a. b.

c.

Isol

euci

ne

Thym

idin

e

Urid

ine

NAG

Acet

ate

0

10

20

30

40

50

60

% In

hibi

tion

10' 30' 60'

Isol

euci

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e

Urid

ine

NAG

Acet

ate

0

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Figure 2 Macromolecular synthesis inhibition in S.aureus ATCC 25923. (a) Profile of compound 1 at a concentration of compound 0.5 µg/ml. (b) Profile of Linezolid at a concentration of 2 µg/ml. (c) Protein synthesis inhibition (%) in S. aureus by compound 1 and Linezolid.

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both staphylococcus and streptococcus infections. Additional pharmacokinetic and pharmacodynamic (PKPD) studies will help in exploring the possibility of taking this compound into preclinical development as a potential candidate against skin and soft tissue infection, pneumonia and bacteremia.

ACKNOWLEDGEMENTS The financial support for research from Daiichi Sankyo India

Pharma Private Limited is acknowledged.

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Figure 3 Predicted binding mode of compound 1 in the active site of E.coli 50S ribosome sub-unit. Compound 1 is shown as a ball & stick model (carbon atoms in green color) and the ribosome sub-unit residues are shown as a thin stick model. The nitrogen atom of the oxazolidinone ring makes a hydrogen bond interaction with the 5’-oxygen atom of G2505 as shown by the yellow dotted line.

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Khera MK, Mathur T, Barman TK, Ramkumar G, Kumar M, et al. (2017) Antibacterial Activity of a Novel 1,2,4-Triazolo [4,3-A] Pyrimidine Oxazolidinone against Broad Spectrum of Gram Positive Pathogens and Molecular Modeling Studies for its Interaction with Ribosome. JSM Microbiology 5(1): 1034.

Cite this article

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