Molecular docking, dynamics and in vitro analysis of multi-target inhibitors for Clostridioides difficile

Abstract

The opportunistic pathogen, Clostridioides difficile owes its extreme pathogenicity for its ability to develop antibiotic resistance and recurrent infections. The current antibiotics used for the treatment are showing declining sensitivity and rising antibiotic resistance. Therefore, it is of interest to develop the anti-clostridial drugs to overcome these issues. Hence, we have explored ZINC library to find the suitable lead compounds against five target proteins of C. difficile. Multistep virtual screening is performed to find the suitable compounds that are checked for their stability using molecular dynamics and are validated in vitro against C. difficile. In our study, five compounds viz., ZINC64969876, ZINC13641164, ZINC13691348, ZINC5554596 and ZINC3894278 that inhibit HisC, Spo0A, PdcA, DAHP synthase and cyclic-di GMP proteins, respectively have been identified. Further, these compounds were tested in vitro against four different isolates of C. difficile and all of them were found to inhibit the pathogen. However, to use these compounds as anti-clostridial drugs, further testing needs to be done. The selected compounds from our study are reported for the first time as antimicrobial agents against C. difficile.

Keywords: Clostridioides difficile; compounds; dynamics; in silico; in vitro; inhibition; molecular docking; pathogen.

Figure 1

Flowchart depicting the methodology used.

Figure 2

Cartoon representation of all five modeled protein structure (A) sporulation transcription factor (Spo0A), (B) c-di-GMP phosphodiesterase (PdcA), (C) histidinol-phosphate transaminase (HisC), (D) 3-deoxy-7-phosphoheptulonate synthase (DAHP synthase) and ( E) bifunctional diguanylate cyclase/ phosphodiesterase (cyclic-diGMP).

Figure 3

Binding orientation of all five docked complexes. Macromolecule is shown in red color and ligand in green color. (A) Sporulation transcription factor (Spo0A) and ZINC13641164, (B) c-di-GMP phosphodiesterase (PdcA) and ZINC13691348, (C) histidinol-phosphate transaminase (HisC) and ZINC64969876, (D) 3-deoxy-7-phosphoheptulonate synthase (DAHP synthase) and ZINC5554596, (E) bifunctional diguanylate cyclase/ phosphodiesterase (cyclic-diGMP) and ZINC3894278.

Figure 4

RMSD plots for Molecular dynamics studies for all five protein ligand complexes.

Figure 5

Ligand RMSD of ZINC13641164, ZINC13691348, ZINC64969876, ZINC5554596 and ZINC3894278.

Figure 6

RMSF plots for Molecular dynamics studies done for all five protein ligand complexes.

Figure 7

Number of hydrogen bonds formed between protein and their respective ligands during simulation.

Figure 8

Radius of Gyration, Solvent accessible surface area (SASA) and total energy plots for all the 5 complexes over 50 ns of simulation time.

Figure 9

Percentage inhibition of C. difficile at different concentrations of compounds.