In vitro and in silico evaluations of actinomycin X2and actinomycin D as potent anti-tuberculosis agents

Abstract

Background: Multidrug-resistant tuberculosis (MDR-TB) is one of the world's most devastating contagious diseases and is caused by the MDR-Mycobacterium tuberculosis (MDR-Mtb) bacteria. It is therefore essential to identify novel anti-TB drug candidates and target proteins to treat MDR-TB. Here, in vitro and in silico studies were used to investigate the anti-TB potential of two newly sourced actinomycins, actinomycin-X₂ (act-X₂) and actinomycin-D (act-D), from the Streptomyces smyrnaeus strain UKAQ_23 (isolated from the Jubail industrial city of Saudi Arabia).

Methods: The anti-TB activity of the isolated actinomycins was assessed in vitro using the Mtb H37Ra, Mycobacterium bovis (BCG), and Mtb H37Rv bacterial strains, using the Microplate Alamar Blue Assay (MABA) method. In silico molecular docking studies were conducted using sixteen anti-TB drug target proteins using the AutoDock Vina 1.1.2 tool. The molecular dynamics (MD) simulations for both actinomycins were then performed with the most suitable target proteins, using the GROningen MAchine For Chemical Simulations (GROMACS) simulation software (GROMACS 2020.4), with the Chemistry at HARvard Macromolecular Mechanics 36m (CHARMM36m) forcefield for proteins and the CHARMM General Force Field (CGenFF) for ligands.

Results: In vitro results for the Mtb H37Ra, BCG, and Mtb H37Rv strains showed that act-X₂ had minimum inhibitory concentration (MIC) values of 1.56 ± 0.0, 1.56 ± 0.0, and 2.64 ± 0.07 µg/mL and act-D had MIC values of 1.56 ± 0.0, 1.56 ± 0.0, and 1.80 ± 0.24 µg/mL respectively. The in silico molecular docking results showed that protein kinase PknB was the preferred target for both actinomycins, while KasA and pantothenate synthetase were the least preferred targets for act-X₂and act-D respectively. The molecular dynamics (MD) results demonstrated that act-X₂ and act-D remained stable inside the binding region of PknB throughout the simulation period. The MM/GBSA (Molecular Mechanics/Generalized Born Surface Area) binding energy calculations showed that act-X₂ was more potent than act-D.

Conclusion: In conclusion, our results suggest that both actinomycins X₂ and D are highly potent anti-TB drug candidates. We show that act-X₂is better able to antagonistically interact with the protein kinase PknB target than act-D, and thus has more potential as a new anti-TB drug candidate.

Keywords: Actinomycin; Anti-TB drug; Molecular docking; Protein kinase PknB; Streptomyces.

Figure 1. In vitro anti-TB activity of isolated act-X₂ and act-D.

Figure 2. Docked compounds act-X₂, shown as sticks in cyan color (A); and act-D, shown as sticks in magenta color (B) in the binding pocket of mycobacterial protein kinase PknB.

Intermolecular interactions are depicted as broken lines.

Figure 3. Mycobacterial protein kinase PknB, shown as a surface in a grey color to highlight the binding cavity.

Docked compounds act-X₂ and act-D are shown as sticks in cyan and magenta color, respectively.

Figure 4. Various trajectory analyses for protein kinase (PknB) complexed with actinomycin D (black lines) and actinomycin X₂ (red lines):

(A) RMSD_protein, (B) RMSD_ligand, (C) Center-of-mass distance between protein and ligand, (D) Protein’s radius of gyration, and (E) RMSF. RMSD_protein, Rg, and RMSF have been calculated using ‘C-alpha’ atoms in the R program using the Bio3D module, while CoM was calculated using gmx distance in Gromacs.

Figure 5. Trajectory analyses for protein kinase (PknB) complexed with act-D (black lines) and act-X₂ (red lines): (A–B) Number of hydrogen-bonds formed between the protein and the act-D and act-X₂, (C) Radial distribution functions of act-D and act-X₂ around PknB, (D) partial density of proteins (solid lines) and ligands (dashed lines), and (E) Molecular Mechanics/Generalized-Born Surface Area protein-ligand binding energy calculated over an interval of 1 ns.