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

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.

Antiprotozoal Activity of Thymoquinone (2-Isopropyl-5-methyl-1,4-benzoquinone) for the Treatment of Leishmania major-Induced Leishmaniasis: In Silico and In Vitro Studies.

Leishmaniasis, a neglected tropical parasitic disease (NTPD), is caused by various Leishmania species. It transmits through the bites of the sandfly. The parasite is evolving resistance to commonly prescribed antileishmanial drugs; thus, there is an urgent need to discover novel antileishmanial drugs to combat drug-resistant leishmaniasis. Thymoquinone (2-isopropyl-5-methyl-1,4-benzoquinone; TQ), a primary pharmacologically active ingredient of Nigella sativa (black seed) essential oil, has been reported to possess significant antiparasitic activity. Therefore, the present study was designed to investigate the in vitro and in silico antileishmanial activity of TQ against various infectious stages of Leishmania major (L. major), i.e., promastigotes and amastigotes, and its cytotoxicity against mice macrophages. In silico molecular dockings of TQ were also performed with multiple selected target proteins of L. major, and the most preferred antileishmanial drug target protein was subjected to in silico molecular dynamics (MD) simulation. The in vitro antileishmanial activity of TQ revealed that the half-maximal effective concentration (EC50), half-maximal cytotoxic concentration (CC50), and selectivity index (SI) values for promastigotes are 2.62 ± 0.12 µM, 29.54 ± 0.07 µM, and 11.27, while for the amastigotes, they are 17.52 ± 0.15 µM, 29.54 ± 0.07 µM, and 1.69, respectively. The molecular docking studies revealed that squalene monooxygenase is the most preferred antileishmanial drug target protein for TQ, whereas triosephosphate isomerase is the least preferred. The MD simulation revealed that TQ remained stable in the binding pocket throughout the simulation. Additionally, the binding energy calculations using Molecular Mechanics Generalized-Born Surface Area (MMGBSA) indicated that TQ is a moderate binder. Thus, the current study shows that TQ is a promising antileishmanial drug candidate that could be used to treat existing drug-resistant leishmaniasis.

In Vitro and In Silico Approaches for the Evaluation of Antimicrobial Activity, Time-Kill Kinetics, and Anti-Biofilm Potential of Thymoquinone (2-Methyl-5-propan-2-ylcyclohexa-2,5-diene-1,4-dione) against Selected Human Pathogens.

Thymoquinone (2-methyl-5-propan-2-ylcyclohexa-2,5-diene-1,4-dione; TQ), a principal bioactive phytoconstituent of Nigella sativa essential oil, has been reported to have high antimicrobial potential. Thus, the current study evaluated TQ's antimicrobial potential against a range of selected human pathogens using in vitro assays, including time-kill kinetics and anti-biofilm activity. In silico molecular docking of TQ against several antimicrobial target proteins and a detailed intermolecular interaction analysis was performed, including binding energies and docking feasibility. Of the tested bacteria and fungi, S. epidermidis ATCC 12228 and Candida albicans ATCC 10231 were the most susceptible to TQ, with 50.3 ± 0.3 mm and 21.1 ± 0.1 mm zones of inhibition, respectively. Minimum inhibitory concentration (MIC) values of TQ are in the range of 12.5-50 µg/mL, while minimum biocidal concentration (MBC) values are in the range of 25-100 µg/mL against the tested organisms. Time-kill kinetics of TQ revealed that the killing time for the tested bacteria is in the range of 1-6 h with the MBC of TQ. Anti-biofilm activity results demonstrate that the minimum biofilm inhibitory concentration (MBIC) values of TQ are in the range of 25-50 µg/mL, while the minimum biofilm eradication concentration (MBEC) values are in the range of 25-100 µg/mL, for the tested bacteria. In silico molecular docking studies revealed four preferred antibacterial and antifungal target proteins for TQ: D-alanyl-D-alanine synthetase (Ddl) from Thermus thermophilus, transcriptional regulator qacR from Staphylococcus aureus, N-myristoyltransferase from Candida albicans, and NADPH-dependent D-xylose reductase from Candida tenuis. In contrast, the nitroreductase family protein from Bacillus cereus and spore coat polysaccharide biosynthesis protein from Bacillus subtilis and UDP-N-acetylglucosamine pyrophosphorylase from Aspergillus fumigatus are the least preferred antibacterial and antifungal target proteins for TQ, respectively. Molecular dynamics (MD) simulations revealed that TQ could bind to all four target proteins, with Ddl and NADPH-dependent D-xylose reductase being the most efficient. Our findings corroborate TQ's high antimicrobial potential, suggesting it may be a promising drug candidate for multi-drug resistant (MDR) pathogens, notably Gram-positive bacteria and Candida albicans.

In Vitro and In Silico Approaches for the Antileishmanial Activity Evaluations of Actinomycins Isolated from Novel Streptomyces smyrnaeus Strain UKAQ_23.

Leishmaniasis, a Neglected Tropical Parasitic Disease (NTPD), is induced by several Leishmania species and is disseminated through sandfly (Lutzomyia longipalpis) bites. The parasite has developed resistance to currently prescribed antileishmanial drugs, and it has become pertinent to the search for new antileishmanial agents. The current study aimed to investigate the in vitro and in silico antileishmanial activity of two newly sourced actinomycins, X2 and D, produced by the novel Streptomyces smyrnaeus strain UKAQ_23. The antileishmanial activity conducted on promastigotes and amastigotes of Leishmania major showed actinomycin X2 having half-maximal effective concentrations (EC50), at 2.10 ± 0.10 µg/mL and 0.10 ± 0.0 µg/mL, and selectivity index (SI) values of 0.048 and 1, respectively, while the actinomycin D exhibited EC50 at 1.90 ± 0.10 µg/mL and 0.10 ± 0.0 µg/mL, and SI values of 0.052 and 1. The molecular docking studies demonstrated squalene synthase as the most favorable antileishmanial target protein for both the actinomycins X2 and D, while the xanthine phosphoribosyltransferase was the least favorable target protein. The molecular dynamics simulations confirmed that both the actinomycins remained stable in the binding pocket during the simulations. Furthermore, the MMPBSA (Molecular Mechanics Poisson-Boltzmann Surface Area) binding energy calculations established that the actinomycin X2 is a better binder than the actinomycin D. In conclusion, both actinomycins X2 and D from Streptomyces smyrnaeus strain UKAQ_23 are promising antileishmanial drug candidates and have strong potential to be used for treating the currently drug-resistant leishmaniasis.