ABSTRACT
Mycobacterium avium complex (MAC) is a serious disease that is mainly caused by infection with the non-tuberculous mycobacteria (NTM), Mycobacterium avium and Mycobacterium intracellulare. Seven new compounds, designated mavintramycins A–G (1–7), were isolated along with structurally related compounds, including amicetin (9) and plicacetin (10), from the culture broth of Streptomyces sp. OPMA40551 as anti-MAC compounds that were active against M. avium and M. intracellulare. Among them, mavintramycin A showed the most potent and selective inhibition of M. avium and M. intracellulare. Furthermore, mavintramycin A was active against more than 40 clinically isolated M. avium, including multidrug-resistant strains, and inhibited the growth of M. avium in a persistent infection cell model using THP-1 macrophages. Mavintramycin A also exhibited in vivo efficacy in silkworm and mouse infection assays with NTM. An experiment to elucidate its mechanism of action revealed that mavintramycin A inhibits protein synthesis by binding to 23S ribosomal RNA in NTM. Mavintramycin A, with a different chemical structure from those of clinically used agents, is a promising drug candidate for the treatment of MAC infectious disease.
KEYWORDS: antibiotic, mavintramycin, Mycobacterium avium complex, infectious disease, non-tuberculous mycobacteria
INTRODUCTION
The prevalence of pulmonary diseases caused by non-tuberculous mycobacteria (NTM) is increasing worldwide, even in immunocompetent individuals. NTM pulmonary disease is an emerging public health issue. In several countries, including the United States, Canada, and Japan, the incidence of NTM diseases is higher than that of tuberculosis (TB) (1–3). Timmins estimated 20,000–30,000, 4,000–5,000, and 35,000–45,000 new patients per year in the US, EU, and Japan, respectively (4). The main causative agents of NTM diseases are Mycobacterium avium and Mycobacterium intracellulare (Mycobacterium avium complex, MAC) in more than 80% of patients with NTM diseases (5). Multiple drug therapy with a macrolide (clarithromycin, CAM, or azithromycin), ethambutol (EB), and rifampicin (RFP) is recommended. However, optimal therapeutic regimens have not yet been established and effective agents are lacking (6, 7). Therefore, the discovery of novel drugs for the treatment of NTM infectious diseases is urgently awaited.
Based on this background, we focused on the discovery of new compounds from microbial resources that exhibited anti-mycobacterial activities against M. avium and M. intracellulare. We reported the effectiveness and usefulness of the MS network-based indexing approach in the screening for new antibiotics (8). Approximately 6,600 microbial culture samples were screened, and the most unique sample derived from the marine actinomycete strain Streptomyces sp. OPMA40551 was selected. Seven new compounds, designated mavintramycins A–G (1–7), were isolated from the culture broth together with four known and structurally related compounds, an unnamed pyrimidine nucleoside (8) (9), amicetin (9) (10), plicacetin (10) (11), and cytosamine (11) (12) (Fig. 1A).
Fig 1.
Results of an MS network-based indexing approach and the structure of the compound obtained from Streptomyces sp. OPMA40551. (A) Structures of compounds 1–11. New compounds (1–7) and known compounds (8–11). (B) One of the molecular network plots including mavintramycins from Streptomyces sp. OPMA40551. Each node represents one consensus MS/MS spectrum-labeled parent mass. Edge thickness indicates similarity in the cosine score. The red color indicates nodes related to metabolites produced by the actinomycete strain selected in our MS network-based screening. The blue color indicates nodes shared with other strains. These data were generated using a cosine similarity score of 0.7 and parent mass tolerance of ±2.0 Da.
In the present study, we report the discovery of new antibiotics, mavintramycins from the culture broth of an actinomycete strain, anti-NTM activity both in vitro and in vivo, and the underlying mechanism of action (MOA).
RESULTS
Screening for anti-MAC antibiotics and discovery of mavintramycins
Among the 6,600 microbial culture samples, 41 cultures exhibited anti-mycobacterial activities against M. avium and M. intracellulare (anti-MAC activity). These cultures were subjected to LC-MS/MS and an MS network-based indexing approach was performed as previously reported (8). Among the 41 cultures, Streptomyces sp. OPMA40551 was selected as the strain producing the highest number of compounds unmatched with known compounds (approximately 7,000 compounds). In the molecular network plot (Fig. 1B), a number of unmatched compounds, including mavintramycins, and related known compounds were observed in the culture of Streptomyces sp. OPMA40551. After fermentation, extraction with ethyl acetate and a two-step chromatographic approach were employed to purify active compounds. This activity-guided purification led to the discovery of new mavintramycins and related known compounds (Fig. 1A). The structures of mavintramycins were elucidated by spectral analyses including various NMR measurements (see supplemental material). These compounds consisted of common cytosine, amosamine, and amicetose moieties and a diverse R moiety (Fig. 1A). Based on the results of the molecular network, this actinomycete produced a cluster containing approximately 20 pyrimidine nucleosides, of which seven compounds, including three mavintramycins (1–7) and four known compounds (8–11), were identified.
Antimicrobial activities of mavintramycins
The anti-mycobacterial activities of mavintramycins A–G (1–7) and related known compounds (8–10) against M. avium JCM15430, M. intracellulare JCM6384, Mycobacterium smegmatis MC2155 Pasteur, and Mycobacterium bovis BCG (standard strains) were evaluated according to a liquid microdilution method (13, 14) (Table 1). These compounds exhibited anti-NTM activity with minimum inhibitory concentrations (MICs) of 0.39–50 μg/mL. Among the 10 compounds examined, mavintramycin A (1) exhibited the most potent anti-MAC activity against M. avium and M. intracellulare with the lowest MICs of 0.78 and 0.39 µg/mL, respectively. Importantly, compound exhibited stronger anti-mycobacterial activities than the clinically used anti-MAC agent, EB.
TABLE 1.
Anti-mycobacterial activities of compounds 1–10 and clinically used drugs
| MIC (μg/mL) | |||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | CAM | RFP | EB | |
| M. avium JCM15430 | 0.78 | 6.25 | >50 | 25 | 12.5 | 3.12 | 3.12 | 6.25 | 3.12 | 3.12 | 0.19 | 0.78 | 12.5 |
| M. intracellulare JCM6384 | 0.39 | 3.12 | 25 | 6.25 | 12.5 | 0.78 | 0.78 | 3.12 | 3.12 | 3.12 | 0.02 | 0.01 | 3.12 |
| M. smegmatis MC2155 Pasteur | 3.12 | 12.5 | >50 | 25 | 50 | 3.12 | 12.5 | 3.12 | 1.56 | 3.12 | 15.6 | 1.56 | 0.78 |
| M. bovis BCG | 1.56 | 6.25 | 6.25 | 6.25 | 6.25 | 1.56 | 0.39 | 0.39 | 0.39 | 0.78 | 0.12 | 0.01 | 1.56 |
The order of the potency of anti-MAC activity was 1 > 6 = 7 >9 = 10 >2 = 8 >4 = 5 >3. Recently known compound amicetin (9) was reprofiled as a promising anti-tuberculosis drug (15), but intriguingly compound 1 was found to be more potent anti-MAC activity than compound 9. These data indicated that the presence of p-aminobenzoic acid (PABA) (5, 9, and 10) at the R position was not essential for anti-MAC activity and that a structural difference at R markedly affected activity.
Inhibition of clinically isolated M. avium by mavintramycin A
The anti-MAC activity of compound 1 was tested against 40 M. avium strains clinically isolated in Japanese hospitals, which included CAM-, RFP-, EB-, and multidrug-resistant (MDR) strains (Table 2). Under these conditions, the MIC of compound 1 against standard M. avium ATCC700898 (wild strain) was 3.12 µg/mL. As listed in Table 2, compound 1 also showed anti-mycobacterial activities against the majority of clinically isolated M. avium strains (MICs 1.0–8.0 µg/mL) and was found to be active against MDR strains (strains 18 and 20) with MICs of 8.0–32 µg/mL.
TABLE 2.
Anti-mycobacterial activity of compound 1 against clinical isolates of M. avium
| Clinical isolate | Resistance to | MIC (μg/mL) | |||
|---|---|---|---|---|---|
| 1 | CAM | RFP | EB | ||
| M. avium 1 | None | 4.0 | 1.0 | 4.0 | 4.0 |
| M. avium 2 | CAM and EB | 8.0 | >64 | 2.0 | >64 |
| M. avium 3 | CAM and EB | 16 | 64 | 4.0 | >64 |
| M. avium 4 | None | 8.0 | 2.0 | 2.0 | 8.0 |
| M. avium 5 | CAM | 4.0 | >64 | 4.0 | 4.0 |
| M. avium 6 | CAM | 8.0 | >64 | 2.0 | 16 |
| M. avium 7 | CAM | 16 | >64 | 4.0 | 32 |
| M. avium 8 | None | 8.0 | 2.0 | 8.0 | 8.0 |
| M. avium 9 | CAM | 4.0 | >64 | 16 | 16 |
| M. avium 10 | CAM and EB | 16 | >64 | 16 | 64 |
| M. avium 11 | CAM | 2.0 | 64 | 2.0 | 16 |
| M. avium 12 | CAM and EB | 8.0 | >64 | 16 | >64 |
| M. avium 13 | CAM and EB | 32 | >64 | 16 | 64 |
| M. avium 14 | CAM | 2.0 | >64 | 4.0 | 16 |
| M. avium 15 | CAM | 4.0 | >64 | 1.0 | 16 |
| M. avium 16 | None | 8.0 | 2.0 | 4.0 | 16 |
| M. avium 17 | CAM and EB | 8.0 | >64 | 8.0 | 32 |
| M. avium 18 | CAM, RFP, and EB | 8.0 | >64 | 32 | >64 |
| M. avium 19 | CAM | 16 | >64 | 16 | 16 |
| M. avium 20 | CAM, RFP, and EB | 32 | >64 | >64 | >64 |
| M. avium 21 | None | 1.0 | 0.25 | 0.063 | 4.0 |
| M. avium 22 | None | 4.0 | 1.0 | 1.0 | 4.0 |
| M. avium 23 | None | 1.0 | 0.5 | 0.016 | 2.0 |
| M. avium 24 | CAM | 1.0 | 32 | 0.031 | 4.0 |
| M. avium 25 | CAM and EB | 1.0 | >64 | 0.5 | 32 |
| M. avium 26 | None | 4.0 | 1.0 | 1.0 | 16 |
| M. avium 27 | None | 2.0 | 0.25 | 1.0 | 4.0 |
| M. avium 28 | None | 2.0 | 0.5 | 0.031 | 8.0 |
| M. avium 29 | None | 1.0 | 0.25 | 0.063 | 2.0 |
| M. avium 30 | CAM and RFP | 1.0 | >64 | >64 | 4.0 |
| M. avium 31 | None | 2.0 | 0.5 | 0.063 | 8.0 |
| M. avium 32 | None | 2.0 | 0.5 | 0.063 | 4.0 |
| M. avium 33 | CAM | 2.0 | 64 | 0.0078 | 4.0 |
| M. avium 34 | None | 2.0 | 8.0 | 0.063 | 16 |
| M. avium 35 | None | 4.0 | 1.0 | 0.031 | 16 |
| M. avium 36 | CAM and EB | 1.0 | 64 | 0.063 | 32 |
| M. avium 37 | CAM | 2.0 | 64 | 0.063 | 8.0 |
| M. avium 38 | CAM | 1.0 | 32 | 0.0039 | 8.0 |
| M. avium 39 | None | 1.0 | 8.0 | 0.13 | 8.0 |
| M. avium 40 | None | 1.0 | 0.13 | 0.13 | 16 |
| M. avium ATCC700898 | Wild strain | 3.12 | 0.39 | 0.04 | 6.25 |
Antimicrobial spectrum of mavintramycin A
Table 3 summarizes the antibacterial and antifungal activities of compounds 1 and 9. Compound 9 was previously reported to be active against Gram-positive bacteria (10), which was confirmed since compound 9 only exhibited moderate activity against Gram-positive Bacillus subtilis and Staphylococcus aureus (Table 3). On the other hand, compound 1 did not exhibit any antimicrobial activity against Gram-positive, Gram-negative (Pseudomonas aeruginosa and Escherichia coli), or fungal strains (Candida albicans and Aspergillus fumigatus). It is important to note that compound 1 was active against M. tuberculosis with an MIC of 0.38 µg/mL. Therefore, compound 1 is a highly selective antibiotic against NTMs and M. tuberculosis. Furthermore, compound 1 did not exhibit cytotoxicity on the THP-1 cells even at 10 µM after 24-h treatment (Table 3).
TABLE 3.
Antibacterial, antifungal, and cytotoxic activities of compounds 1 and 9a
| Test microorganism | MIC (μg/mL) | |
|---|---|---|
| 1 | 9 | |
| Mycobacterium avium ATCC700898 | 3.12 | 6.25 |
| Mycobacterium tuberculosis H37Rv | 0.38 | N.T. |
| Aspergillus fumigatus NBRC33022 | >32 | >32 |
| Candida albicans ATCC90029 | >32 | >32 |
| Bacillus subtilis PCI219 | >50 | 25 |
| Pseudomonas aeruginosa IFO12689 | >50 | >50 |
| Escherichia coli JM109 | >50 | >50 |
| Staphylococcus aureus FDA209P | >50 | 25 |
| Test cell line | IC50 (μg/mL) | |
| THP-1 cells (before differentiation) | >10 | >10 |
| THP-1 cells (after differentiation) | >10 | >10 |
N.T., not tested.
Inhibition of M. avium infection in THP-1 macrophages by mavintramycin A
Mycobacteria survive in the macrophages of MAC patients, and infection persists for a long time. Therefore, the antibacterial activities of drugs against intracellular mycobacteria need to be examined. In this cell-based model, an infection assay of M. avium in THP-1 macrophages was performed according to a previously reported method (16) with some modifications (see supplemental material). Under these conditions, the colony number of M. avium in THP-1 macrophages gradually decreased day by day (day 0 vs day 4, left two columns in Fig. 2). M. avium persisted [8.3 × 103 colony-forming unit (CFU)/mL] in control cells on day 4. Under the same condition, intracellular M. avium on day 4 significantly decreased to 31% of the control in the presence of compound 1 (10 μg/mL). RFP (10 µg/mL) potently inhibited intracellular M. avium infection (22% of the control), whereas CAM and EB exhibited moderate (74%) and no effect (105%) on the intracellular persistent infection, respectively (Fig. 2).
Fig 2.
Efficacy of compound 1 against intracellular M. avium in THP-1 macrophages. THP-1 macrophages infected with M. avium ATCC700898 (day 0) were incubated with a drug (compound 1, CAM, RFP, or EB at 10 µg/mL each) at 37°C. On day 4, cells were disrupted, diluted, and plated to count the CFUs of surviving M. avium. Bars represent the means of CFU counts, and error bars represent the standard deviation. Data are expressed as mean values ± SD. Asterisks indicate a significant difference from the control with *P < 0.05 or **P < 0.001 (Tukey’s Honestly Significant Difference test).
Time-kill effect of mavintramycin A
To investigate whether compound 1 exerts bactericidal or bacteriostatic effects, a time-kill assay of compound 1 against M. avium was performed according to a previously reported method (17) with some modifications. As shown in Fig. 3A through C, M. avium gradually grew for 13 days (control). Under these conditions, compound 1 inhibited growth (OD600) in time- and dose-dependent manners. In the presence of compound 1 even at an MIC dose (3.12 µg/mL), M. avium stopped growing after day 4, resulting in 50% inhibition on day 13. At a dose of 10-fold MIC (31.2 µg/mL), the growth of M. avium was completely inhibited after day 10, indicating that compound 1 exerted bactericidal effects against M. avium. In the presence of CAM at a dose of 10-fold MIC (3.9 µg/mL), the growth of M. avium was inhibited until day 7; however, very fast regrowth subsequently occurred (Fig. 3B and C), resulting in higher CFU on day 13 than the control (Fig. 3D). In the presence of EB at the 10-fold MIC dose (62.5 µg/mL), the growth of M. avium was inhibited after day 4, resulting in 40% inhibition of the control on day 13 (Fig. 3B). Importantly, in the combination of CAM (10-fold MIC, 3.9 µg/mL) with compound 1 (MIC, 3.12 µg/mL), the growth of M. avium was rapidly suppressed after day 2 and marked inhibition continued over time (until at least day 13, Fig. 3C), resulting in 0.01% of control CFU (1 × 1010 CFU/mL) on day 13 (Fig. 3D).
Fig 3.
Time-kill curve of compound 1 against M. avium. (A) Compound 1 was added at the following doses: 0 (control, ○), 2- (◆), 5- (♦), and 10- (■) fold MIC to the M. avium ATCC700898 culture (OD600 = 0.05–0.1 on day 0). M. avium was incubated at 37°C and growth was measured at OD600 over time. (B) 1 (■), CAM (▲), and EB (●) were added at a dose of 10-fold of each MIC to the M. avium ATCC700898 culture (adjusted OD600 = 0.05–0.1). M. avium was incubated at 37°C. Growth was measured at OD600. (C) Compound 1 at MIC (3.12 µg/mL, ◆) was added in combination with 10-fold MIC of CAM [CAM alone (▲), combination (■)]. (D) Ten microliters of the culture broth of panel C after 13 days were serially diluted 10-fold and plated onto 7H10 agar plates to count CFUs.
Combination of mavintramycin A with clinically used drugs
Since the combination of compound 1 with CAM showed effectiveness against M. avium in the time-kill assay, detailed experiments were done to investigate the combination effect of compound 1 with a clinically used anti-NTM drug (CAM, RFP, and EB) by the checkerboard assay (18). From the result, as summarized in Table 4, a combination of compound 1 with CAM or RFP exhibited an additive effect against M. avium, while a combination with EB was found to be indifferent. For these results, it was suggested that compound 1 be suitably combined with RFP and CAM for the treatment of MAC infection.
TABLE 4.
MICs of compound 1 and an anti-NTM drug, alone and together, against M. avium ATCC700898
| Drugs | MIC (μg/mL) | FIC | FICI | Outcome | ||||
|---|---|---|---|---|---|---|---|---|
| Alone | In combination | |||||||
| 1 | Drug | 1 | Drug | 1 | Drug | |||
| RFP | 3.12 | 0.05 | 0.39 | 0.024 | 0.13 | 0.48 | 0.61 | Additive |
| CAM | 0.39 | 0.78 | 0.195 | 0.25 | 0.50 | 0.75 | Additive | |
| EB | 6.25 | 3.12 | 6.25 | 1.00 | 1.00 | 2.00 | Indifferent | |
FICI, fractional inhibitory concentration index.
MOA of mavintramycin A
To elucidate the underlying MOA, we initially investigated the incorporation of radio-labeled precursors into macromolecules (DNA, RNA, proteins, and peptidoglycans) in mycobacteria. M. smegmatis was used in this experiment because M. avium cannot grow in the minimum medium. M. smegmatis was incubated with 3H-thymidine, 3H-L-leucine, 3H-uracil, or 3H-N-acetyl-D-glucosamine for 120 min in the presence of compound 1 (25 μg/mL), and the respective macromolecules were collected to measure radioactivity. As shown in Fig. 4, compound 1 almost completely inhibited the incorporation of 3H-L-leucine into the macromolecule fraction (protein), and this was followed by the strong inhibition of the incorporation of 3H-thymidine into the DNA fraction. Only moderate inhibition of the incorporation of 3H-uracil and 3H-N-acetyl-D-glucosamine was observed (Fig. 4).
Fig 4.
Effects of compound 1 on macromolecule synthesis in M. smegmatis M341. Precursor uptake using M. smegmatis. The incorporation of the precursors, 3H-thymidine (A), 3H-uracil (B), 3H-L-leucine (C), and 3H-N-acetyl-D-glucosamine (D) into macromolecule fractions in M. smegmatis was measured (see Materials and Methods). ◆, control and ■, compound 1 (25 μg/mL).
In the next MOA experiment, M. avium strains resistant to compound 1 were isolated, and the causative genes responsible for resistance were identified. When wild-type M. avium ATCC700898 was incubated on agar medium containing compound 1, even at a dose of 6.25 µg/mL (twice MIC), no colonies were obtained. Therefore, resistant strains were initially isolated at a very low concentration of compound 1 (0.39 μg/mL), and the concentration of compound 1 then gradually increased (0.78, 1.56, 3.12, 6.24, and 12.5 µg/mL) to finally isolate resistant strains. Table S4 summarizes the MIC values of the three resistant strains (MA1–MA3) and wild-type (WT) strain. The genome sequences of MA1–MA3 and WT were elucidated, and the mutation sites were analyzed. As summarized in Table 5, all eight mutation points were found in a genome named MAV101_007745 encoding 23S ribosomal RNA. Among them, only one mutation site (1992T > G) was common in all resistant strains. Structurally related compound 9 was previously reported to bind to 23S ribosomal RNA (19). Importantly, MA1–MA3 were also resistant to compound 9 at 50 µg/mL. Thus, we concluded that compound 1 primarily inhibited protein synthesis by binding to 23S ribosomal RNA, which had a negative impact on the synthesis of DNA.
TABLE 5.
Genome sequencing results of MA1–MA3 and WT
| Chromosome | Position | Gene name | Mutation | Product | Observed MAs |
|---|---|---|---|---|---|
| Contig042 | 112,916 | MAV101_007745 | 1992T > G | 23S ribosomal RNA | MA1–MA3 |
| 112,073 | 1149G > T | MA1 and MA3 | |||
| 112,073 | 1149G > C | MA1 and MA3 | |||
| 112,138 | 1214A > G | MA1 and MA3 | |||
| 112,138 | 1214A > T | MA1 and MA3 | |||
| 112,146 | 1222A > C | MA1 and MA3 | |||
| 112,155 | 1231T > A | MA1 and MA3 | |||
| 112,155 | 1231T > C | MA1 and MA3 |
In vivo activity of mavintramycin A in mycobacterial infection
Efficacy of silkworm infection by M. smegmatis
We previously established a silkworm infection assay with M. smegmatis (13, 14, 20). M. smegmatis is a non-pathogenic bacterium. However, when M. smegmatis was injected into silkworms at a higher cell number (1 × 107 CFU/larva) and the injected silkworms were incubated at a higher temperature (37°C), they all died within 40–50 h. In addition, clinically used anti-TB drugs, such as isoniazid, RFP, and EB, exerted therapeutic effects in this silkworm assay (20). Therefore, we assessed compound 1 in this assay. After being infected with M. smegmatis (n = 5), all silkworms died within 43 h (Fig. 5A). Under this condition, compound 1 exerted dose-dependent therapeutic effects in this assay, and the half-life (38 h) of infected silkworms was significantly prolonged to 50 h at the highest dose of compound 1 (50 µg/larva, Fig. 5A). Furthermore, the 50% effective dose (ED50) values of these compounds 43 h after infection were 4.25 µg/larva (Fig. 5), indicating the better efficacy of compound 1 than EB (Fig. 5B, ED50, 35.4 µg/larva). In addition, compound 1 did not exhibit toxicity against silkworms for at least 60 h.
Fig 5.
In vivo efficacy of compound 1 using silkworms. (A) Therapeutic effects of compound 1 in the silkworm infection assay with M. smegmatis. Dose: ▼ 3.12, ● 12.5, and ▲ 50 µg/larva for 1. ◇, control (no drug) and □, no infection with M. smegmatis. (B) Therapeutic effects of EB in the silkworm infection assay with M. smegmatis. Dose: ▼ 3.12, ● 12.5, and ▲ 50 µg/larva for 1. ◇ control (no drug) and □, no infection with M. smegmatis.
Efficacy of mouse infection by M. avium
Compound 1 was evaluated in a mouse infection assay with M. avium. The tolerability of compound 1 was confirmed by the repeated intravenous and oral administration of 30 mg/kg of body weight for 2 weeks in mice. When compound 1 was intravenously (10 mg/kg of body weight) or orally (100 mg/kg of body weight) administrated in mice, compound 1 disappeared in 2 h after intravenous administration, and almost compound 1 was not absorbed after oral administration. Furthermore, compound 1 was not detected in the lungs of mice with both administrations. Also, the administration dose was determined to be 10 mg/kg of body weight/day because of the highest solubility of MMA at that time and in vitro MIC data. Finally, as the first in vivo efficacy experiment, compound 1 was assessed by its nasal administration (10 mg/kg of body weight/day) in a mouse infection assay with M. avium. Mice were intratracheally infected with M. avium (1.0 × 106 CFU/body), and M. avium (3.9 × 105 CFU/lung) was recovered from the right lungs after 7 days. Under this condition, the nasal administration of compound 1 (10 mg/kg of body weight/day) for 7 days resulted in a significant reduction in M. avium (2.2 × 105 CFU/lung). Amikacin (AMK) (100 mg/kg of body weight/day) also suppressed lung infection to 0.7 × 105 CFU/lung (Fig. 6). Therefore, compound 1 was active in vivo against M. avium in mouse lungs.
Fig 6.
In vivo efficacy of compound 1 using mice. Effects of compound 1 (10 mg/kg of body weight) and AMK (100 mg/kg of body weight) on colony-forming units in the right lung of M. avium JCM15430-infected mice (n = 8). Data are expressed as mean values ± SD. Asterisks indicate a significant difference from the vehicle with P < 0.05 (Tukey’s Honestly Significant Difference test).
DISCUSSION
In the present study, approximately 6,600 microbial culture broth samples were screened for new anti-MAC compounds using M. avium and M. intracellulare. With the effective help of an MS networking approach (8), we selected marine-derived Streptomyces sp. OPMA40551 as the most promising strain, which produced new anti-MAC compounds. The culture broth was predicted to potentially contain 20 structurally related compounds, which formed a cluster on the MS network plot (Fig. 1B). Among them, 11 compounds were isolated, and their structures were elucidated to yield seven new mavintramycins (1–7) and four known compounds, including amicetin (9) and plicacetin (10) (Fig. 1A). The compounds in this cluster had the same scaffold consisting of cytosine, amosamine, and amicetose. We initially filed mavintramycins in a patent in 2017 (21), and Aryal and coworkers (22) very recently discovered compound 1 (previously named OPMA40551F) during an amicetin biosynthetic study. The structural elucidation of mavintramycins, including compound 1, was fully described in this study for the first time (supplemental material).
A number of nucleoside antibiotics have been isolated from actinomycetes. For example, blasticidin S isolated from Streptomyces griseochromogenes in 1958 exhibited broad antifungal and antimicrobial activities (23), while amicetin (9) and plicacetin (10) (co-purified with mavintramycins in this study), originally isolated from Streptomyces plicatus in 1953 (10), exhibited antimicrobial activity against Gram-positive bacteria, including M. tuberculosis (11). Amicetin has recently been attracting increasing attention as a new lead for the treatment of MDR TB and TB-HIV co-infection (15). In the present study, amicetin (9) and plicacetin (10) also exhibited anti-MAC activity, and mavintramycin A (1), with a simpler structure than compounds 9 and 10, exhibited the most potent activity among the 11 compounds isolated from Streptomyces sp. OPMA40551 (Table 1). Bu et al. (24) reported that the related known compound, streptcytosine, exhibited the same anti-NTM activity as compound 1. In that study, only streptcytosine A, which has the same cytosine, amosamine, amicetose, and PABA moieties as amicetin, exhibited anti-NTM activity, while streptcytosine B to E, which did not have amosamine or PABA moieties, did not. These results suggest that the amosamine and/or PABA moieties are essential for anti-mycobacterial activity; however, only the amosamine moiety appears to be essential for anti-NTM activity. Furthermore, compound 1 exhibited promising in vitro activities against MAC; (i) compound 1 was highly selective against NTM and TB, even among Gram-positive bacteria (Table 3), (ii) it was non-toxic against five animal cell lines tested at 10–50 µg/mL (Table 3), (iii) it was active against all clinically isolated M. avium, including MDR strains (Table 2), (iv) compound 1 was bactericidal against M. avium (Fig. 3A), (v) it was able to block the re-growth of CAM-resistant M. avium at an MIC dose (Fig. 3C), and (vi) it was effective in a MAC persistent infection model in macrophages (Fig. 2).
Among the precursors incorporated into corresponding macromolecules, compound 1 completely inhibited protein synthesis and strongly inhibited DNA synthesis in M. smegmatis, while it exerted very weak effects on RNA and peptidoglycan syntheses (Fig. 4). Structurally related amicetin also showed a similar inhibition pattern in macromolecule synthesis (25). As described above, rapidly growing M. smegmatis was used for this experiment because slowly growing M. avium cannot grow to incorporate precursors into macromolecules in the minimum medium in a short time (120 min). To further investigate the MOA, we attempted to isolate compound 1-resistant M. avium. In the presence of compound 1 at an MIC dose or higher, resistant strains did not emerge. Therefore, we started to isolate them at a low dose of compound 1 (×1/16, 1/8, 1/4, and 1/2 MIC doses) and gradually increased the dose. As a result, three compound 1-resistant strains (MA1–MA3) with MICs of 6.25–12.5 µg/mL were obtained. This result implies that natural compound 1-resistant MAC hardly emerged. In the genomic analysis of the three resistant strains, the same point mutation was defined at 23S ribosomal RNA in M. avium (Table 5). Furthermore, MA1–MA3 were found to be resistant to amicetin at 50 µg/mL. Therefore, we concluded that compound 1 inhibited protein synthesis by binding to ribosomal RNA. The MOA of amicetin was extensively examined by several groups (15, 19, 26). Serrano et al. (26) demonstrated the binding of amicetin to the 70S ribosomal subunit of Thermus thermophilus by crystallography and revealed that amicetin occupied the peptidyl transferase center P-site of ribosomes. Accordingly, compound 1 was plausible to bind to the corresponding site of M. avium. Furthermore, compound 1 is considered to bind to a distinct site on the ribosome from clinically used CAM (macrolide) and amikacin (AMK) (aminoglycoside), corroborating the result that no cross-resistant M. avium strains with compound 1 were observed (Table 2). The precise binding site of compound 1 is currently being investigated.
These biological characteristics prompted us to perform another in vivo experiment on compound 1 for its development as a novel anti-MAC drug. The results obtained showed that compound 1 was effective in both the silkworm infection assay with M. smegmatis (Fig. 5) and the mouse infection assay with M. avium (Fig. 6). In the mouse assay, compound 1 was nasally administered to infected mice in order to reach the lungs.
One reason for the long-term treatment of MAC disease is the inadequate efficacy of clinically used drugs. The activity of CAM disappeared after day 7 in the time-kill assay, and the efficacy of CAM was low in a MAC-persistent infection in THP-1 macrophages. To compensate for these conditions, Arikayce, an amikacin liposome inhalation suspension, was recently approved as a new drug. However, the achievement rate of a shortened treatment period was only 29% (27), which indicates that MAC disease is an unmet medical need. Another cause is the emergence of drug-resistant bacteria. Multidrug-resistant MAC against CAM, RFP, and EB, which are first-line drugs, have already been reported (6). A combination with Arikayce has not resolved this MDR issue because AMK-resistant MAC strains have already been reported (28). These causes are attributed to all clinically used drugs, including Arikayce, being developed by drug repositioning. To overcome this issue, mavintramycin A (1) is a promising new antibiotic for the treatment of MAC disease. A goal of MAC treatment is to establish new drug regimens containing the best combination of new drugs that will shorten the duration of MAC therapy and combat drug-resistant MAC. Further evaluations of compound 1 are warranted. Mavintramycin A (1) may ultimately be useful for the treatment of TB as well as MAC in combination with conventional MAC and TB drugs or with promising candidates that are also currently under development.
MATERIALS AND METHODS
Materials
Mavintramycins A–G (1–7) and four known compounds (8–11) were purified from a culture broth of actinomycetes in our laboratory. CAM, RFP, AMK, imipenem, and ciprofloxacin were purchased from Wako Pure Chemical Industries (Osaka, Japan). Middlebrook 7H9 broth (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) containing 0.05% Tween 80 (Tokyo Chemical Industries, Tokyo, Japan) and 10% albumin dextrose catalase enrichment [5% bovine serum albumin, Sigma Aldrich (MO, USA); 2% glucose, Wako Pure Chemical Industries (Osaka, Japan); and 0.85% NaCl, Wako Pure Chemical Industries] was used for the cultivation of mycobacteria. Third-molting larval stage silkworms, Bombyx mori (Hu·yo × Tukuba·Ne), were purchased from Ehime Sansyu (Ehime, Japan). Silk Mate 2S, an artificial diet containing antibiotics, was purchased from Nosan Corporation (Kanagawa, Japan). The following mycobacterial strains were used in the present study: M. avium JCM15430, M. avium ATCC700898, M. intracellulare JCM6384, M. bovis BCG Pasteur, M. smegmatis MC2155, and M. smegmatis M341. M. bovis BCG Pasteur, M. smegmatis MC2155, and M. smegmatis M341 were laboratory strains. M. avium JCM15430 and M. intracellulare JCM6384 were purchased from the Riken BioResource Research Center (Ibaraki, Japan). M. avium ATCC700898 was purchased from the American Type Culture Collection (VA, USA).
Assay for anti-mycobacterial activity
Anti-mycobacterial activities against these strains were evaluated by the liquid microdilution method according to a previously established procedure (13, 14).
Checkerboard microtiter assay
To investigate whether mavintramycin A (1) exhibits synergistic activity against M. avium ATCC700898 in combination with a clinically used anti-NTM drug (CAM, RFP, and EB), a checkerboard microtiter assay was performed by the established method (18). The fractional inhibitory concentration index (FICI) was calculated from the following formula: FICI = (MIC drug A in combination/MIC drug A alone) + (MIC drug B in combination/MIC drug B alone). The FICI was interpreted as indicating a synergistic effect (FICI ≤ 0.5), an additive effect (0.5 < FICI ≤ 1.0), indifference (1.0 ≤ FICI < 4.0), and an antagonistic effect (4.0 ≤ FICI).
Isolation of compounds 1–11
The culture broth (3.0 L, pH 8.1) of Streptomyces sp. OPMA40551 was extracted with ethyl acetate (twice, 3.0 L each). The organic layer was dried over Na2SO4 and concentrated under a reduced pressure to give a yellow material (164.4 mg). Crude materials were dissolved in a small amount of MeOH, applied to an octadecyl silyl column (8.2 g), and eluted stepwise with 0%, 20%, 40%, 60%, 70%, 80%, and 100% MeOH (60 mL each). The 70% MeOH fraction containing compounds 1, 3, 4, 5, 6, and 8 was concentrated under a reduced pressure to give a pale-yellow material (21.5 mg). These compounds were finally purified by preparative high-performance liquid chromatography (HPLC) under the following conditions: column, PEGASIL ODS SP100 (i.d. 20 × 250 mm); mobile phase, 20%–45% CH3CN (gradient 40 min) containing 0.05% TFA; detection, ultraviolet (UV) at 210 nm; and flow rate, 6.0 mL/min. Under these conditions, compounds 1, 3, 4, 5, 6, and 8 were eluted as a peak with retention times of 26, 25, 27, 31, 34, and 25 min, respectively. Each fraction was concentrated using a freeze dryer to give pure compounds 1 (4.6 mg, white powder), 4 (1.6 mg, white powder), 5 (2.0 mg, white powder), 6 (0.9 mg, white powder), and a mixture of 3 and 8 (2.7 mg, white powder). Compounds 3 and 8 were purified by HPLC under the following conditions: column, PEGASIL ODS SP100 (i.d. 20 × 250 mm); mobile phase, 21% CH3CN isocratic containing 0.05% TFA; detection, UV at 210 nm; and flow rate, 6.0 mL/min. Compounds 3 (1.7 mg, 42 min, white powder) and 8 (1.6 mg, 45 min, white powder) were obtained.
Compound 2 (0.8 mg, white powder) was obtained from the 80% MeOH fraction, and compounds 7 (2.9 mg, white powder), 9 (23.3 mg, pale-yellow powder), 10 (4.8 mg, white powder), and 11 (8.2 mg, white powder) were obtained from the 60% MeOH fraction. These compounds were finally purified by preparative HPLC under the following conditions: column, PEGASIL ODS SP100 (i.d. 20 × 250 mm); mobile phase, 30%–60% CH3CN (gradient 40 min) containing 0.05% TFA for the 80% MeOH fraction, 8%–50% CH3CN (gradient 50 min) containing 0.05% TFA for 60% MeOH fraction; detection, UV at 210 nm; and flow rate, 6.0 mL/min. Under these conditions, compound 2 was eluted as a peak with a retention time of 20 min in the 80% MeOH fraction, and compounds 7, 9, 10, and 11 were eluted as a peak with retention times of 30, 23, 28, and 14 min, respectively, in the 60% MeOH fraction.
The fermentation of Streptomyces sp. OPMA40551, physicochemical properties, and structural elucidation of compounds 1–7 are described in the supplemental material.
Time-kill curve analysis
Precultures of M. avium ATCC700898 were diluted to an OD600 of 0.1 in 4 mL of 7H9 medium in test tubes. Test compounds were added at 1-, 2-, 5-, and 10-fold MIC to M. avium ATCC700898, incubated at 37°C, and OD600 was measured over time.
Isolation of MMA-resistant M. avium ATCC700898
M. avium ATCC700898 was inoculated on ×2 MIC-containing 7H10 agar medium, incubated at 37°C, and no colonies were obtained. Therefore, resistant M. avium were obtained by gradually increasing the concentration of compound 1 from the concentration below MIC. The addition of ×1/16, 1/8, 1/4, and 1/2 MIC of compound 1 to a 24-well plate was followed by 1 mL of M. avium ATCC700898 (4 × 106 CFU/mL) in 7H9 medium and an incubation at 37°C for approximately 7 days. After the incubation, 50 µL of each well was transferred into 1 mL of 7H9 medium with ×1/4 MIC of compound 1 and incubated at 37°C for approximately 7 days. Similarly, the concentration of compound 1 was gradually increased and the incubation was repeated up to ×8 MIC of compound 1. After the incubation, 100 µL of the 100-fold diluted culture was plated on 7H10 agar medium and incubated at 37°C for 10 days, and resistant M. avium colonies (MA1, MA2, and MA3) were obtained. A genome analysis of the resistant M. avium obtained (MA1, MA2, and MA3) was outsourced to Hokkaido System Science Co., Ltd (Hokkaido, Japan).
Precursor uptake assay
Precultures of M. smegmatis M341 were diluted to an OD600 of 0.2 in 4 mL of 7H9 medium in test tubes. The radiated precursors [methyl-3H] thymidine (0.9 µCi/mL, 5.5 pmol), [5,6-3H] Uracil (0.15 µCi/mL, 21.4 pmol), [4,5-3H] L-leucine (0.15 µCi/mL, 5.5 pmol), and [glucosamine-6-3H] N-acetyl-D-glucosamine (0.15 µCi/mL, 64.3 pmol) as well as compound 1 (final 25 µg/mL) were added and incubated at 37°C at 150 rpm. After 15, 30, 60, and 120 min 1 mL of the culture was removed from each tube, and 10% trichloroacetic acid (TCA) was added. After cooling on ice for 1 h, suction filtration was performed with a glass fiber filter (Whatman GF/B 24 mm Φ), the filter was washed with 2% TCA and 95% EtOH, and radioactivity (DPM) was measured with a liquid scintillation counter.
Silkworm infection assay with M. smegmatis
The silkworm infection assay with M. smegmatis was performed according to a previously established method (13, 14, 20). Hatched silkworm larvae (Ehime Sansyu, Ehime, Japan) were raised by feeding Silk Mate 2S (Nihon Nosan Kogyo, Kanagawa, Japan) in an incubator at 27°C until the fourth molting stage. On the first day of fifth-instar larvae, silkworms (n = 5) were fed Silk Mate 2S. On the second day, M. smegmatis (2.5 × 107 CFU/larva in 50 µL of Middlebrook 7H9 broth) was injected into the hemolymph through the dorsal surface of the silkworm using a disposable 1 mL syringe with a 27-G needle (Terumo, Tokyo, Japan). Compound 1 or EB solubilized in 50 µL of 10% dimethyl sulfoxide (DMSO) was injected into the hemolymph within 1 h of infection with M. smegmatis. All M. smegmatis-infected silkworms died within 43 h when no sample was administered. After sample injection, the number of silkworms that survived was counted at the indicated time until 70 h. The survival rate at the indicated dose of each sample was calculated when all M. smegmatis-infected silkworms without the sample injection had died (43 h). ED50 values (43 h after infection) were calculated according to a previous method (13, 14, 20).
Mouse infection assay with M. avium
This in vivo evaluation was basically performed according to the method reported by Shiozawa et al. (29). In brief, female BALB/c mice (Japan SLC, 7 weeks old) were intratracheally infected with M. avium JCM15430 (106 CFU/0.05 mL/mouse) (day 0). After being infected with M. avium, drugs (compound 1 at 10 mg/kg of body weight in 20 µL of water or AMK at 100 mg/kg of body weight in 20 µL of water) were intranasally administered to mice (n = 8) daily for 6 days (days 1–6). On day 7 (24 h after the final drug treatment), the right lungs were harvested and homogenized in saline (1 mL) to quantify bacterial numbers (CFU). Homogenates (serial 1:10, 1:100, 1:1,000, and 1:10,000 dilutions) were plated on nutrient 7H11 agar plates, and CFUs were assessed after a 2-week incubation at 37°C. The left lungs were stored for pathological analysis.
Statistical analyses
Experimental data are expressed as mean ± standard deviation of the mean, and statistically significant differences among treatments were detected by one-way ANOVA (P < 0.05) using GraphPad Prism Software (GraphPad Software, Inc., La Jolla, CA, USA). When ANOVA results were statistically significant, individual treatment comparisons were made using Tukey’s post-test analysis. For the silkworm infection assay, outcomes were determined using the Kaplan-Meier method, and statistical analysis was performed using the log-rank test (GraphPad Prism Software, Inc., La Jolla, CA, USA).
ACKNOWLEDGMENTS
We express our thanks to Dr. Kenichiro Nagai and Ms. Noriko Sato, School of Pharmacy, Kitasato University, for the measurements of mass spectra and NMR, respectively.
This work was supported by the Japan Agency for Medical Research and Development (AMED) under Grant Number JP20nk0101375h0004 and 21fk0108429h0001 (to H.T.), and The Uehara Memorial Foundation Grant (to H.T.), and the Japan Society for the Promotion of Science KAKENHI Grant Number 15K07867 (to N.K.) from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and the Kitasato University Research Grant for Young Researchers (to K.H.).
N.K. and H.T. designed the research. T.N. and N.H. collected clinically isolated M. avium strains. A.K. collected microbial samples. K.H. and S.S. performed experiments. K.H., N.K., S.S., T.O., and H.T. wrote the manuscript. All authors approved the final version of the manuscript.
K.H., N.K, A.K., and H.T. are named as inventors on patent applications related to this work: patent #PCT/JP2018/032048.
Contributor Information
Hiroshi Tomoda, Email: tomodah@pharm.kitasato-u.ac.jp.
Cesar A. Arias, Houston Methodist Academic Institute, Houston, Texas, USA
ETHICS APPROVAL
The mouse infection assay with M. avium was outsourced to LSI Medience Corporation (Tokyo, Japan) and approved by the Animal Care Committees of LSI Medience Corporation.
SUPPLEMENTAL MATERIAL
The following material is available online at https://doi.org/10.1128/aac.00917-23.
Supplemental figures, tables, and text.
ASM does not own the copyrights to Supplemental Material that may be linked to, or accessed through, an article. The authors have granted ASM a non-exclusive, world-wide license to publish the Supplemental Material files. Please contact the corresponding author directly for reuse.
REFERENCES
- 1. Adjemian J, Olivier KN, Seitz AE, Holland SM, Prevots DR. 2012. Prevalence of nontuberculous mycobacterial lung disease in U.S. Medicare beneficiaries. Am J Respir Crit Care Med 185:881–886. doi: 10.1164/rccm.201111-2016OC [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2. Marras TK, Mendelson D, Marchand-Austin A, May K, Jamieson FB. 2013. Pulmonary nontuberculous mycobacterial disease, Ontario, Canada, 1998-2010. Emerg Infect Dis 19:1889–1891. doi: 10.3201/eid1911.130737 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3. Namkoong H, Kurashima A, Morimoto K, Hoshino Y, Hasegawa N, Ato M, Mitarai S. 2016. Epidemiology of pulmonary nontuberculous mycobacterial disease, Japan. Emerg Infect Dis 22:1116–1117. doi: 10.3201/eid2206.151086 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4. Timmins GS. 2020. What are the challenges in commercial non-tuberculous mycobacteria (NTM) drug discovery and how should we move forward? Expert Opin Drug Discov 15:7–9. doi: 10.1080/17460441.2020.1673362 [DOI] [PubMed] [Google Scholar]
- 5. Busatto C, Vianna JS, da Silva LV, Ramis IB, da Silva PEA. 2019. Mycobacterium avium: an overview. Tuberculosis (Edinb) 114:127–134. doi: 10.1016/j.tube.2018.12.004 [DOI] [PubMed] [Google Scholar]
- 6. Griffith DE, Aksamit T, Brown-Elliott BA, Catanzaro A, Daley C, Gordin F, Holland SM, Horsburgh R, Huitt G, Iademarco MF, Iseman M, Olivier K, Ruoss S, von Reyn CF, Wallace RJ, Winthrop K, ATS Mycobacterial Diseases Subcommittee, American Thoracic Society, Infectious Disease Society of America . 2007. An official ATS/IDSA statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases. Am J Respir Crit Care Med 175:367–416. doi: 10.1164/rccm.200604-571ST [DOI] [PubMed] [Google Scholar]
- 7. Adelman MH, Addrizzo-Harris DJ. 2018. Management of nontuberculous mycobacterial pulmonary disease. Curr Opin Pulm Med 24:212–219. doi: 10.1097/MCP.0000000000000473 [DOI] [PubMed] [Google Scholar]
- 8. Koyama N, Tomoda H. 2019. MS network-based screening for new antibiotics discovery. J Antibiot 72:54–56. doi: 10.1038/s41429-018-0109-0 [DOI] [PubMed] [Google Scholar]
- 9. Kate SDG AS, Sonawave S, Periyasamy G. 2013. Nucleoside analogue as an anticancer compound. WO2013144894
- 10. DeBoer C, Caron EL, Hinman JW. 1953. Amicetin, a new streptomyces antibiotics. J Am Chem Soc 75:499–500. doi: 10.1021/ja01098a518 [DOI] [Google Scholar]
- 11. Haskell TH, Ryder A, Frohardt RP, Fusari SA, Jakubowski ZL, Bartz QR. 1958. The isolation and characterization of three crystalline antibiotics from Streptomyces plicatus. J Am Chem Soc 80:743–747. doi: 10.1021/ja01536a055 [DOI] [Google Scholar]
- 12. P. Sensi AMG, Gallo GG, Roland G. 1957. Isolation and structure determination of a new amicetin-like antibiotic: amicetin B. Antibiot Chemother 12:645–652. [PubMed] [Google Scholar]
- 13. Hosoda K, Koyama N, Kanamoto A, Tomoda H. 2019. Discovery of nosiheptide, griseoviridin, and etamycin as potent anti-mycobacterial agents against Mycobacterium avium complex. Molecules 24:1495. doi: 10.3390/molecules24081495 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14. Hosoda K, Koyama N, Hamamoto H, Yagi A, Uchida R, Kanamoto A, Tomoda H. 2020. Evaluation of anti-mycobacterial compounds in a silkworm infection model with Mycobacteroides abscessus. Molecules 25:4971. doi: 10.3390/molecules25214971 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15. Nelli MR, Heitmeier KN, Looper RE. 2021. Dissecting the nucleoside antibiotics as universal translation inhibitors. Acc Chem Res 54:2798–2811. doi: 10.1021/acs.accounts.1c00221 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16. Rose SJ, Neville ME, Gupta R, Bermudez LE. 2014. Delivery of aerosolized liposomal amikacin as a novel approach for the treatment of nontuberculous mycobacteria in an experimental model of pulmonary infection. PLoS One 9:e108703. doi: 10.1371/journal.pone.0108703 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17. Pryjma M, Burian J, Thompson CJ. 2018. Rifabutin acts in synergy and is bactericidal with frontline Mycobacterium abscessus antibiotics clarithromycin and tigecycline, suggesting a potent treatment combination. Antimicrob Agents Chemother 62:e00283-18. doi: 10.1128/AAC.00283-18 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18. Kaushik A, Ammerman NC, Tasneen R, Story-Roller E, Dooley KE, Dorman SE, Nuermberger EL, Lamichhane G. 2017. In vitro and in vivo activity of biapenem against drug-susceptible and rifampicin-resistant Mycobacterium tuberculosis. J Antimicrob Chemother 72:2320–2325. doi: 10.1093/jac/dkx152 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19. Shammas C, Donarski JA, Ramesh V. 2007. NMR structure of the peptidyl transferase RNA inhibitor antibiotic amicetin. Magn Reson Chem 45:133–141. doi: 10.1002/mrc.1932 [DOI] [PubMed] [Google Scholar]
- 20. Yagi A, Uchida R, Hamamoto H, Sekimizu K, Kimura K, Tomoda H. 2017. Anti-Mycobacterium activity of microbial peptides in a silkworm infection model with Mycobacterium smegmatis. J Antibiot 70:685–690. doi: 10.1038/ja.2017.23 [DOI] [PubMed] [Google Scholar]
- 21. Tomoda H, Koyama N, Kanamoto A, Hashimoto J, Kozone I.. 2019. Novel compound having therapeutic activity against Mycobacterium avium complex infection, and method for producing same. WO2019044941.
- 22. Aryal N, Chen J, Bhattarai K, Hennrich O, Handayani I, Kramer M, Straetener J, Wommer T, Berscheid A, Peter S, Reiling N, Brötz-Oesterhelt H, Geibel C, Lämmerhofer M, Mast Y, Gross H. 2022. High plasticity of the amicetin biosynthetic pathway in Streptomyces sp. SHP 22-7 led to the discovery of streptcytosine P and cytosaminomycins F and G and facilitated the production of 12F-plicacetin. J Nat Prod 85:530–539. doi: 10.1021/acs.jnatprod.1c01051 [DOI] [PubMed] [Google Scholar]
- 23. Takeuchi S, Hirayama K, Ueda K, Sakai H, Yonehara H. 1958. Blasticidin S, a new antibiotic. J Antibiot 11:1–5. [PubMed] [Google Scholar]
- 24. Bu YY, Yamazaki H, Ukai K, Namikoshi M. 2014. Anti-mycobacterial nucleoside antibiotics from a marine-derived Streptomyces sp. TPU1236A. Mar Drugs 12:6102–6112. doi: 10.3390/md12126102 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25. Bloch A, Coutsogeorgopoulos C. 1966. Inhibition of protein synthesis by amicetin, a nucleoside antibiotic. Biochemistry 5:3345–3351. doi: 10.1021/bi00874a038 [DOI] [PubMed] [Google Scholar]
- 26. Serrano CM, Kanna Reddy HR, Eiler D, Koch M, Tresco BIC, Barrows LR, VanderLinden RT, Testa CA, Sebahar PR, Looper RE. 2020. Unifying the aminohexopyranose- and peptidyl-nucleoside antibiotics: implications for antibiotic design. Angew Chem Int Ed Engl 59:11330–11333. doi: 10.1002/anie.202003094 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27. Anonymous . 2018. Study to evaluate efficacy of LAI when added to multi-drug regimen compared to multi-drug regimen alone (CONVERT). NCT02344004
- 28. Kim SY, Kim DH, Moon SM, Song JY, Huh HJ, Lee NY, Shin SJ, Koh WJ, Jhun BW. 2021. Association between 16S rRNA gene mutations and susceptibility to amikacin in Mycobacterium avium complex and Mycobacterium abscessus clinical isolates. Sci Rep 11:6108. doi: 10.1038/s41598-021-85721-5 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29. Shiozawa A, Kajiwara C, Ishii Y, Tateda K. 2020. N-acetyl-cysteine mediates protection against Mycobacterium avium through induction of human beta-defensin-2 in a mouse lung infection model. Microbes Infect 22:567–575. doi: 10.1016/j.micinf.2020.08.003 [DOI] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Supplemental figures, tables, and text.