Exploring Indonesian actinomycete extracts for anti-tubercular compounds: Integrating inhibition assessment, genomic analysis, and prediction of its target by molecular docking

Abstract

Tuberculosis (TB) is the foremost cause of infectious fatality globally. The primary global challenge in combatting TB lies in addressing the emergence of drug-resistant variants of the disease. However, the number of newly approved agents for treating TB has remained remarkably low over recent decades. Hence, research endeavors for discovering novel anti-TB agents are always needed. In the present study, we screened over 1,500 culture extracts from actinomycetes isolated in Indonesia for their inhibitory activity against Mycobacterium smegmatis used as a surrogate in the primary screening. The initial screening yielded approximately 6.2 % hit extracts, with a selection criterion of >80 % growth inhibition. The confirmed hit extracts were subsequently subjected to growth inhibition assay against Mycobacterium bovis and Mycobacterium tuberculosis. Approximately 20 % of the hit extracts that showed growth inhibition also exhibited efficacy against M. bovis BCG and M. tuberculosis H37Rv pathogenic strain. An active compound was successfully purified from a large-scale culture of the most potent representative extract by high-performance liquid chromatography and thin-layer chromatography. The structure of the active compound was elucidated by mass spectrometry and nuclear magnetic resonance. This compound displayed structural similarities to actinomycin group and exhibited robust inhibition, with IC₅₀ values of 0.74, 0.02, and 0.07 μg/mL against M. smegmatis, M. bovis, and M. tuberculosis, respectively. The Actinomycetes strain A612, which produced the active compound, was taxonomically classified by phylogenetic analysis of 16s rRNA gene and whole genome sequencing data as Streptomyces parvus. Computational genome analysis utilizing anti-SMASH 7.0 unveiled that S. parvus A612 strain harbors 40 biosynthetic gene clusters with the potential to produce 16 known (with >70 % similarity) and 24 unknown compounds. A non-ribosomal peptide synthesis (NRPS) gene cluster associated with actinomycin D biosynthesis was also identified, boasting an 85 % similarity. Molecular docking analysis of actinomycin D and 21 potential M. tuberculosis targets revealed possible interactions with multiple targets. The purified active compound inhibited recombinant M. tuberculosis shikimate kinase (MtSK), which validated the results obtained from the docking analysis.

Keywords: Actinomycetes; Active compound; Docking study; Extracts; Screening; Shikimate kinase; Tuberculosis; Whole genome.

Fig. 1

Performance metrics of actinomycetes extracts screening against M. smegmatis. Quality control values of all twenty 96-well assay plates in the primary screening of the extracts (1,534 extracts). The values of the S/B ratio (SB) and Z′ are shown (A). A plot showing percentage inhibition of anti-mycobacterial activity by all extracts in the primary assay (B). Inhibition level and hit rate calculation from primary screening (C).

Fig. 2

A screening cascade of actinomycetes extracts against Mycobacterium. The number of technical replicate assays is indicated (n). Individual steps of the cascade are described in the text.

Fig. 3

Evaluation of purification process and purity of active compound. The TLC pattern of the pure compound observed with cerium sulfate stain (right side), UV wavelength 254 nm (middle) and 366 nm (left) with eluent dichloromethane: methanol (20 : 1). A red bracket indicating the specific location of the targeted pure active compound. (A). The quantification of the purity of the active compound was performed using HPLC. The active peak is indicated by the blue arrow, and it exhibited a purity of 97.2 % (B). UV visible spectrum absorbance of active compound after purification (C).

Fig. 4

Mass Spectra (A), 1H NMR (B) and 13C NMR (C) of purified compounds from strain A612.

Fig. 5

Dose-dependent inhibition for IC50 determination of active compound inhibited M. smegmatis(A), M. bovis(B) and M. tuberculosis(C).

Fig. 6

The phylogenetic tree and morphology of strain A612. Phylogenetic tree derived from 16S rRNA gene sequences of strain A612 and members of the genus Streptomyces, reconstructed with the neighbor-joining method. The 16S rRNA gene sequence of Kitasatospora acidiphila MMS16-CNU292 was used as the outgroup. Bootstrap values (>50 %) based on 1000 replicates are shown at branch nodes. Bar, 0.005 Knuc substitutions per nucleotide position (A). The morphology of A612 on Yeast Starch Agar after incubation for 5 days. The presence of dissolved pigment in the agar medium was observed (B).

Fig. 7

Whole-genome sequence-based tree from strain A612 and relative genome sequences. The branch lengths are scaled in terms of genome BLAST distance phylogeny (GBDP) distance formula d5 (Meier-Kolthoff and Göker, 2019). Numbers above branches are GBDP pseudo-bootstrap support values > 60 % from 100 replications.

Fig. 8

Distribution of subsystems from Streptomyces A612 strain annotated using rapid annotations subsystem technology (RAST) server (A) and A comparison of actinomycin gene clusters of Streptomyces A612 and Streptomyces anulatus(B). For figure A, the heat maps bar displayed in a grid, each row represents the similarity based on color and bar size by considering species cluster, subspecies cluster, percent G + C content, delta statistic, genome size, and protein count. Similar bar size and color indicated similar genome feature.

Fig. 9

Docked compound F5(23) in the various M. tuberculosis protein. 3D (A-E) and 2D (F-J) interaction are presented. Enoyl reductase (A and F), Coenzyme A biosynthesis bifunctional protein (B and G), Aspartate-semialdehyde dehydrogenase (C and H), Adenosylmethionine-8-amino-7-oxononanoate aminotransferase (D and I) and Antigen 85 complex (E and J). The yellow circle highlights the specific position where the compound has been docked within the protein.

Fig. 10

The docking of compound F5(23) into the M. tuberculosis shikimate kinase (MtSK) enzyme. The 3D and 2D visualizations for two binding sites within MtSK are presented. The first site (A and C) is where ATP typically binds naturally, and the second site (B and D) is where shikimate natively docks. The yellow circle is used to emphasize the precise location where the compound has been docked within the protein.