Elsevier

Microbial Pathogenesis

Volume 147, October 2020, 104205
Microbial Pathogenesis

Computer aided ligand based screening for identification of promising molecules against enzymes involved in peptidoglycan biosynthetic pathway from Acinetobacter baumannii

https://doi.org/10.1016/j.micpath.2020.104205Get rights and content

Highlights

  • A.baumannii has been considered as Priority-I as suggested by the WHO.

  • In-house library was prepared using a similarity search of a known inhibitor.

  • Computer-aided ligand-based virtual screening approach was performed.

  • Identification of promising molecules against Mur family proteins.

Abstract

A. baumannii has been considered as Priority-I as suggested by the World Health Organization (WHO) and the most critical pathogenic microorganism for causing nosocomial infection in imunno-compromised hospital-acquired patients due to multi-drug resistance (MDR). In the current study, we utilized “Computer-aided ligand-based virtual screening approach” for identification of promising molecules against Mur family proteins based on the known inhibitor (Naphthyl Tetronic Acids ((5Z)-3-(4-chlorophenyl)-4-hydroxy-5-(1-naphthylmethylene) furan-2(5H)-one)) of MurB from E. coli. The in-house library was prepared using a similarity search of a known inhibitor (Drug Bank ID: DB07296) against several relevant chemical databases. The molecules obtained from virtual screening of Naphthyl Tetronic Acids in-house library were successively subjected to physicochemical and ADMET screening. After this, the molecules which passed all the filters, subsequently subjected into interaction analysis with the drug target proteins (MurB, MurD, MurE and MurG) of A. baumanni and the results explained that four molecules were promising (CHEMBL468144, DB07296, Enamine_T5956969 and 54723243) for further molecular dynamics simulations. The free and ligand bounded proteins that undergone MD simulation are listed as follows: MurB, MurB-CHEMBL468144, MurB-DB07296, MurE, MurE-54723243, MurE-DB07296, MurD, MurD-Enamine_T5956969, MurD-DB07296, MurG, MurG-CHEMBL468144, and MurG-DB07296. Based on global and essential dynamics analysis, the stability order of molecules towards MurB (CHEMBL468144 > DB07296); MurD (Enamine_T5956969 > DB07296); MurE (54723243 > DB07296) and MurG (CHEMBL468144 > DB07296) indicates that the newly identified molecules are more promising one in comparison with the existing inhibitor. Based on all the docking and MD simulation results, the stability order of the free and ligand bounded protein are as follows; MurB and MurB-ligand complexes > MurD and MurD-ligand complexes > MurG and MurG-ligand complexes > MurE and MurE-ligand complexes. Finally, the selected compounds would be recommended for further experimental investigations and used as promising inhibitors of the infection caused by A. baumannii.

Introduction

A species which have the characteristic of strictly aerophilic and biochemically defined as catalase-positive, indole-negative, oxidase-negative, gram-negative and citrate positive are grouped into the genus of Acinetobacter [1], from these species, Acinetobacter baumannii is an essential representative and clinically the most important [2]. This organism is responsible for the cause of different hospital-acquired nosocomial infections by using its virulence factors. The possible virulence factor of this organism includes cell surface hydrophobicity, outer membrane proteins (OMPs), toxic slime polysaccharides and verotoxins. Cell surface hydrophobicity is a vital element for bacterial sticking together as well as avoiding being phagocytosed among the virulence factors [1,3]. On the other hand, extracellular enzymes (esterases, certain amino-peptidases, acid phosphatases, and toxins) that are producing in the cytosol, as well as other fluid constituents, are factors that play a significant role in the pathogenesis. It is the cause of tissue-related disease, especially in breathing system [1]. In addition to this, A. baumannii has been considered as Priority-I as suggested by WHO and the most critical pathogenic microorganism for causing nosocomial infection in imunno-compromised hospital patients due to MDR [4]. Therefore, it is essential to find out novel anti-microbial agents against their drug target proteins. For this study, we were used three-dimensional models of following Mur family proteins, namely MurB (unpublished), MurD (unpublished), MurE [5] and MurG [6]. In-house library of 48 molecules is prepared based on the similarity with the known inhibitor of MurB from E. coli (Drug Bank ID: DB07296) [7].

Gram-negative cell walls are strong enough to endure turgor pressure, extreme temperatures and pHs, as well as elastic sufficiently to expand several times [8]. In all eubacteria (except Mycoplasma species and a few other species), the murein (peptidoglycan) is the crucial exoskeleton which is required to resist the internal cytoplasmic turgor (osmotic) pressure. It contains oligo (GlcNAc-MurNAc) glycan strands. The strands are cross-linked by short peptides and form a net-like polymeric structure that surrounds the plasma membrane [9]. Mainly, peptidoglycan uses as a structural role in the bacterial cell wall and give structural strength. The osmotic pressure of the cytoplasm is counteracting by peptidoglycan. Particles size around 2 nm can pass through the peptidoglycan in both Gram-positive and Gram-negative bacteria [10]. As mentioned above, peptidoglycan is the main constituent for the synthesis of the cell wall, and which is synthesized with the series of steps or sequential reactions is in the biosynthetic pathway. During the peptidoglycan biosynthetic pathway, UDP-N-acetylglucosamine 1-carboxyvinyltransferase (MurA) facilitates the first step of the reaction. This enzyme involves attacking the electrophilic carbon two positions of phosphoenolpyruvate (PEP) leading to the cleavage of the carbon oxygen bond [11].

Similarly, the substrate obtained from the first reaction (UDP-N-acetylmuramic acid (UDPMurNAc)) is catalyzes using UDP-N-acetylenolpyruvoylglucosamine reductase (MurB) [11,12]. Additionally, MurC, MurD, MurE and MurF are involved for the addition of a short polypeptide chain to the UDPMurNAc which are responsible for the successive additions of l-alanine, d-glutamate, meso-diaminopimelate or l-lysine, and D-alanyl-d-alanine to UDP-N-acetylmuramic acid. All four Mur ligases are structurally similar to one another, even though they display low sequence identity or sequence similarity [5,13,14]. The final step of the peptidoglycan biosynthetic pathway catalyzes with the help of two crucial enzymes, namely MraY (membrane transferase enzyme) and MurG (membrane transferase enzyme). The first enzyme is responsible the transfer of the phospho-N-acetylmuramoyl-pentapeptide motif onto the undecaprenyl phosphate carrier lipid [15] and the second is the transfer of a GlcNAc subunit on undecaprenyl-pyrophosphoryl-MurNAc-pentapeptide (lipid intermediate I) to form undecaprenyl-pyrophosphoryl-MurNAc-(pentapeptide) GlcNAc (lipid intermediate II) [16]. The entire enzyme participated for the biosynthesis of peptidoglycan sequentially inside the bacteria, including A. baumanni, and this pathway is unique for prokaryotes. So, an identification of the enzymes involved in this pathway as a potential drug target and it is essential to counterpart the severity of A. baumannii in hospital patients.

Several classes of inhibitors from natural or synthetic or semi-synthetic sources are inhibitors of drug target proteins of Mur family [17]. Recently, the few small-molecules have been reported as potent and promising inhibitors against Mur enzymes which include MurA [18], MurB [19], MurF [20], MurG [6], MurC, MurD and MurE [5]. Discovery of small-molecule inhibitors against several Mur enzymes has been many restrictions, such as low anti-bacterial activities in cells, poor physicochemical properties and evolving drug resistance bacteria time to time [21,22]. Recently, one of the inhibitors named as Naphthyl Tetronic Acids ((5Z)-3-(4-chlorophenyl)-4-hydroxy-5-(1-naphthylmethylene) furan-2(5H)-one) showing strong activity against Mur family enzyme, particularly MurB from E. coli. This potent compound was synthesized in the laboratory with 3 step process [7]. This compound showed the broad-spectrum activity and inhibits all nine drug target enzymes with excellent binding affinity and minimal inhibitory concentration (MIC) values (specifically with a Kd value of 40 nM and MIC values of 2 μg ml−1 against multiple E. coli strains). According to the pre-clinical trial report, the co-crystal structure of Naphthyl compound with E. coli MurB was determined at a resolution of 2.5 Å (PDB ID: 2Q85) and reported in RCSB - Protein Data Bank [23], and the structure shows crucial interactions between the three-part of the compound named as, the furanone core, the Naphthyl moiety, and the chlorophenyl side chain [7] (Fig. 1). As we know, A. baumannii has MDR ability in nature, and we need to have significant inhibitors based on the Naphthyl Tetronic Acids scaffold using Ligand-Based Virtual Screening (LBVS). Therefore, an identification of the enzymes involved in this pathway as potential drug target and exploring novel inhibitors against them is essential to counterpart the severity of A. baumannii in hospital patients.

In the current study, we utilized the computer-aided ligand-based virtual screening approach for the identification of promising molecules against Mur family proteins based on the known inhibitor of E. coli. The in-house library was prepared using a similarity search of a known inhibitor against several relevant chemical databases. The molecules obtained from virtual screening of Naphthyl Tetronic Acids in-house library were successively subjected to physicochemical and ADMET properties for further screening. Finally, the compounds were selected according to the above screening techniques of each Mur protein from this family. A series of MD simulations were performed for validation of the newly identified putative inhibitors compared with the existing one. Finally, the selected compounds would be recommended for further experimental investigations and used as promising inhibitors of the infection caused by A. baumannii.

Section snippets

Identification of existing inhibitors and preparation of an in-house library of napthyl tetranoic acid scaffold

The known inhibitor of MurB from E. coli was obtained from extensive literature survey using PubMed (https://www.ncbi.nlm.nih.gov/pubmed/) and other literature sources, and this inhibitor is found to be multi-target inhibitors of bacterial peptidoglycan biosynthetic pathways [7]. The known inhibitor belongs to Naphthyl Tetronic Acids family, which includes compounds from 5a, 5b, 5c, 5d, 5e, 5f, 5g, 5h, 5i, 5j, and 5k [7]. The similarity principle is used for LBVS, which infers that similar

Identification of the known inhibitor and preparation of napthyl tetraonic acid in-house library

Recently, Mansour and his collaborators [7] explored the Naphthyl Tetronic Acids family of molecules and used as an inhibitor of bacterial Mur family protein. Naphthyl Tetronic Acids (existing inhibitor) (Drug Bank ID: DB07296) and its derivatives are most promising molecules which had the capability for inhibitions of MurB from E. coli and documented as a potent inhibitor for the first time. Apart from E. coli MurB, Naphthyl Tetronic Acids (existing inhibitor) and its semi-synthetic

Conclusion

As we know, A. baumannii has multi-drug resistance ability in nature, and therefore we need to identify the promising inhibitors against the pathogen. In this study, we utilized computer-aided ligand-based virtual screening based on the Naphthyl Tetronic Acid scaffold. So that, an in-house library was prepared using similarity search of Naphthyl Tetronic Acids against several important chemical databases and screened towards MurB, MurD, MurE and MurG models. Based on the results obtained

Ethical standards

Ethical standards are compulsory for studies relating to human and animal subjects.

Author statement

Amit Kumar Singh: Conceptualization, Gizachew Muluneh Amera, Jayaraman Muthukumaran and Amit Kumar Singh: Methodology, Software, Gizachew Muluneh Amera, Rameez Jabeer Khan, Amita Pathak, Rajat Kumar Jha, Jayaraman Muthukumaran and Amit Kumar Singh: Data curation, Gizachew Muluneh Amera: Writing - Original draft preparation, Gizachew Muluneh Amera, Rameez Jabeer Khan, Jayaraman Muthukumaran, Amit Kumar Singh: Visualization, Investigation, Amit Kumar Singh: Supervision, Gizachew Muluneh Amera,

Declaration of competing interest

None

Acknowledgements

Dr. Amit Kumar Singh thanks the Department of Science and Technology (DST) and Indian National Science Academy (INSA), New Delhi, India. Gizachew Muluneh Amera thanks the College of Natural Science, Wollo University, Dessie, Ethiopia for the sponsorship. The authors also thank to Supercomputing Facility for Bioinformatics & Computational Biology, IIT Delhi.

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