Retinestatin, a Polyol Polyketide from a Termite Nest-Derived Streptomyces sp.

A new polyol polyketide, named retinestatin (1), was obtained and characterized from the culture of a Streptomyces strain, which was isolated from a subterranean nest of the termite Reticulitermes speratus kyushuensis Morimoto. The planar structure of 1 was elucidated on the basis of the cumulative analysis of ultraviolet, infrared, mass spectrometry, and nuclear magnetic resonance spectroscopic data. The absolute configuration of 1 at 12 chiral centers was successfully assigned by employing a J-based configuration analysis in combination with ROESY correlations, a quantum mechanics-based computational approach to calculate NMR chemical shifts, and a 3 min flash esterification by Mosher's reagents followed by NMR analysis. Biological evaluation of retinestatin (1) using an in vitro model of Parkinson's disease revealed that 1 protected SH-SY5Y dopaminergic cells from MPP+-induced cytotoxicity, indicating its neuroprotective effects.

DS86760016, a Leucyl-tRNA Synthetase Inhibitor, Is Active against Mycobacterium abscessus.

Benzoxaboroles are a new class of leucyl-tRNA synthetase inhibitors. Epetraborole, a benzoxaborole, is a clinical candidate developed for Gram-negative infections and has been confirmed to exhibit favorable activity against a well known pulmonary pathogen, Mycobacterium abscessus. However, according to ClinicalTrials.gov, in 2017, a clinical phase II study on the use of epetraborole to treat complicated urinary tract and intra-abdominal infections was terminated due to the rapid emergence of drug resistance during treatment. Nevertheless, epetraborole is in clinical development for nontuberculous mycobacteria (NTM) disease especially for Mycobacterium avium complex-related pulmonary disease (MAC-PD). DS86760016, an epetraborole analog, was further demonstrated to have an improved pharmacokinetic profile, lower plasma clearance, longer plasma half-life, and higher renal excretion than epetraborole in animal models. In this study, DS86760016 was found to be similarly active against M. abscessus in vitro, intracellularly, and in zebrafish infection models with a low mutation frequency. These results expand the diversity of druggable compounds as new benzoxaborole-based candidates for treating M. abscessus diseases.

Tandocyclinones A and B, Ether Bridged C-Glycosyl Benz[a]anthracenes from an Intertidal Zone Streptomyces sp.

Two new proton-deficient metabolites, tandocyclinones A and B (1 and 2), were discovered via the chemical profiling of the Streptomyces sp. strain TDH03, which was isolated from a marine sediment sample collected from the intertidal mudflat in Tando Port, the Republic of Korea. The structures of 1 and 2 were elucidated as new ether-bridged C-glycosyl benz[a]anthracenes by using a combination of spectroscopic analyses of ultraviolet (UV) and mass spectrometry (MS) data, along with nuclear magnetic resonance (NMR) spectra, which were acquired in tetrahydrofuran (THF)-d8 selected after an extensive search for a solvent, resulting in mostly observable exchangeable protons in the 1H NMR spectrum. Their configurations were successfully assigned by applying a J-based configuration analysis, rotating-frame Overhauser enhancement spectroscopy (ROESY) NMR correlations, chemical derivatization methods based on NMR (a modified version of Mosher's method) and circular dichroism (CD) (Snatzke's method using Mo2(OAc)4-induced CD), as well as quantum-mechanics-based computational methods, to calculate the electronic circular dichroism (ECD). Tandocyclinones A and B (1 and 2) were found to have weak antifungal activity against Trichophyton mentagrophytes IFM40996 with an MIC value of 128 µg/mL (244 and 265 µM for 1 and 2, respectively). A further biological evaluation revealed that tandocyclinone A (1) displayed inhibitory activity against Mycobacterium avium (MIC50 = 40.8 µM) and antiproliferative activity against SNU638 and HCT116 cancer cells, with IC50 values of 31.9 µM and 49.4 µM, respectively.

Synergistic Effect of Q203 Combined with PBTZ169 against Mycobacterium tuberculosis.

Q203 is a first-in-class drug candidate against Mycobacterium tuberculosis. In its recently completed phase 2 clinical trial, Q203 reduced the number of live M. tuberculosis cells in a dose-dependent manner. This orally active small molecule blocks M. tuberculosis growth by inhibiting the cytochrome bc1 complex, which consequently inhibits the synthesis of ATP. Here, we studied the interaction profiles of Q203 with several antituberculosis drugs or drug candidates (specifically, bedaquiline, PBTZ169, PA-824, OPC-67683, SQ109, isoniazid, rifampin, streptomycin, and linezolid) using the checkerboard method, based on resazurin microtiter assays (REMAs). In the assay, none of the interactions between Q203 and the tested drugs were antagonistic, and most of the interactions were additive. However, the interaction between Q203 and PBTZ169 was synergistic, with a fractional inhibitory concentration index of 0.5. Furthermore, Q203 (one-half the MIC50) and PBTZ169 (one-half the MIC50) inhibited more bacterial growth on an agar plate compared to the dimethyl sulfoxide (DMSO) control. This synergistic effect was no longer effective when the Q203-PBTZ169 combination was tested against an M. tuberculosis mutant containing a T313A mutation causing resistance to Q203, suggesting that QcrB inhibition is integral to the Q203-PBTZ169 interaction. Thus, this synergy is not an off-target mechanism. Zebrafish (Danio rerio)-Mycobacterium marinum infection and a curing model further validated the synergistic effect of Q203 and PBTZ169 in vivo. In this study, the synergy between these two new antituberculosis drugs, Q203 and PBTZ169, is an important finding that could lead to the development of a new TB regimen.

Gwanakosides A and B, 6-Deoxy-α-l-talopyranose-Bearing Aromatic Metabolites from a Streptomyces sp. and Coculture with Pandoraea sp.

Single-strain cultivation of a mountain soil-derived Streptomyces sp. GA02 and its coculture with Pandoraea sp. GA02N produced two aromatic products, gwanakosides A and B (1 and 2, respectively). Their spectroscopic analysis revealed that 1 is a new dichlorinated naphthalene glycoside and 2 is a pentacyclic aromatic glycoside. The assignment of the two chlorine atoms in 1 was confirmed by the analysis of its band-selective CLIP-HSQMBC spectrum. The sugars in the gwanakosides were identified as 6-deoxy-α-l-talopyranose based on 1H-1H coupling constants, Rotating frame Overhauser enhancement spectroscopy (ROESY) NMR correlations, and chemical derivatization followed by spectroscopic and chromatographic analyses. The absolute configuration of 2, whose production was enhanced approximately 100-fold in coculture, was proposed based on a quantum mechanics-based chemical shift analysis method, DP4 calculations, and the chemically determined configuration of 6-deoxy-α-l-talopyranose. Gwanakoside A displayed inhibitory activity against pathogenic bacteria, including Staphylococcus aureus (MIC = 8 µg/mL) and Mycobacterium tuberculosis (MIC50 = 15 µg/mL), and antiproliferative activity against several human cancer cell lines (IC50 = 5.6-19.4 µM).

Taeanamides A and B, Nonribosomal Lipo-Decapeptides Isolated from an Intertidal-Mudflat-Derived Streptomyces sp.

Two new lipo-decapeptides, namely taeanamides A and B (1 and 2), were discovered from the Gram-positive bacterium Streptomyces sp. AMD43, which was isolated from a mudflat sample from Anmyeondo, Korea. The exact molecular masses of 1 and 2 were revealed by high-resolution mass spectrometry, and the planar structures of 1 and 2 were elucidated using NMR spectroscopy. The absolute configurations of 1 and 2 were determined using a combined analysis of 1H-1H coupling constants and ROESY correlations, the advanced Marfey's method, and bioinformatics. The putative nonribosomal peptide synthetase pathway for the taeanamides was identified by analyzing the full genome sequence data of Streptomyces sp. AMD43. We also found that taeanamide A exhibited mild anti-tuberculosis bioactivity, whereas taeanamide B showed significant bioactivity against several cancer cell lines.

Structures and Biosynthetic Pathway of Coprisamides C and D, 2-Alkenylcinnamic Acid-Containing Peptides from the Gut Bacterium of the Carrion Beetle Silpha perforata.

Coprisamides C and D (1 and 2) were isolated from a gut bacterium, Micromonospora sp. UTJ3, of the carrion beetle Silpha perforata. Based on the combined analysis of UV, MS, and NMR spectral data, the planar structures of 1 and 2 were elucidated to be unreported derivatives of coprisamides A and B, cyclic depsipeptides bearing a 2-alkenylcinnamic acid unit and the unusual amino acids ß-methylaspartic acid and 2,3-diaminopropanoic acid. The absolute configuration of 1 was determined using the advanced Marfey's method, phenylglycine methyl ester derivatization, and J-based configuration analysis. The biosynthetic gene clusters for the coprisamides were investigated based on genomic data from coprisamide-producing strains Micromonospora sp. UTJ3 and Streptomyces sp. SNU533. Coprisamide C (1) was active against the Mycobacterium tuberculosis mc2 6230 strain.

A Screening of the MMV Pandemic Response Box Reveals Epetraborole as a New Potent Inhibitor against Mycobacterium abscessus.

Mycobacterium abscessus is the one of the most feared bacterial respiratory pathogens in the world. Unfortunately, there are many problems with the current M. abscessus therapies available. These problems include misdiagnoses, high drug resistance, poor long-term treatment outcomes, and high costs. Until now, there have only been a few new compounds or drug formulations which are active against M. abscessus, and these are present in preclinical and clinical development only. With that in mind, new and more powerful anti-M. abscessus medicines need to be discovered and developed. In this study, we conducted an in vitro-dual screen against M. abscessus rough (R) and smooth (S) variants using a Pandemic Response Box and identified epetraborole as a new effective candidate for M. abscessus therapy. For further validation, epetraborole showed significant activity against the growth of the M. abscessus wild-type strain, three subspecies, drug-resistant strains and clinical isolates in vitro, while also inhibiting the growth of M. abscessus that reside in macrophages without cytotoxicity. Furthermore, the in vivo efficacy of epetraborole in the zebrafish infection model was greater than that of tigecycline. Thus, we concluded that epetraborole is a potential anti-M. abscessus candidate in the M. abscessus drug search.

Etamycin as a Novel Mycobacterium abscessus Inhibitor.

The increase in drug-resistant Mycobacterium abscessus, which has become resistant to existing standard-of-care agents, is a major concern, and new antibacterial agents are strongly needed. In this study, we introduced etamycin that showed an excellent activity against M. abscessus. We found that etamycin significantly inhibited the growth of M. abscessus wild-type strain, three subspecies, and clinical isolates in vitro and inhibited the growth of M. abscessus that resides in macrophages without cytotoxicity. Furthermore, the in vivo efficacy of etamycin in the zebrafish (Danio rerio) infection model was greater than that of clarithromycin, which is recommended as the core agent for treating M. abscessus infections. Thus, we concluded that etamycin is a potential anti-M. abscessus candidate for further development as a clinical drug candidate.

Rifamycin O, An Alternative Anti-Mycobacterium abscessus Agent.

Mycobacterium abscessus is the most difficult-to-treat nontuberculous mycobacteria because of its resistance to many antibiotics. In this study, we screened the Korea Chemical Bank library for a bioluminescent reporter assay to identify molecules capable of acting against M. abscessus. On application of the assay, rifamycin O showed excellent in vitro activity with a narrow range of the minimum inhibitory concentration required to inhibit the growth of 90% of the bacterium (MIC90 = 4.0-6.2 µM); its in vivo efficacy in the zebrafish (Danio rerio) infection model was comparable to that of rifabutin at 25 µM. Furthermore, rifamycin O did not show significant toxicity in cells and the zebrafish model. These results are the first in vivo indication that rifamycin O may be a drug candidate for treating M. abscessus infections.

Target Engagement and Binding Mode of an Antituberculosis Drug to Its Bacterial Target Deciphered in Whole Living Cells by NMR.

Detailed information on hit-target interaction is very valuable for drug discovery efforts and indispensable for rational hit to lead optimization. We developed a new approach combining NMR in whole-cells in-cell NMR) and docking to characterize hit-target interaction at the atomic level. By using in-cell NMR, we validated target engagement of the antituberculosis imidazopyridine amide (IPA) series with the subunit b of the cytochrome bc1:aa3, the major respiratory terminal oxidase in mycobacteria. The most advanced IPA called Q203 is currently in clinical trial. Using its derivative IPA317, we identified the atoms of the drug interacting with the cytochrome b in whole cells. NMR data and the self-organizing map algorithm were used to cluster a large set of drug-target complex models. The selected ensemble revealed IPA317 in a transient cavity of the cytochrome b, interacting directly with the residue T313, which is the site of spontaneous mutation conferring resistance to the IPA series. Our approach constitutes a pipeline to obtain atomic information on hit-target interactions in the cellular context.

PPAR-α Activation Mediates Innate Host Defense through Induction of TFEB and Lipid Catabolism.

The role of peroxisome proliferator-activated receptor α (PPAR-α) in innate host defense is largely unknown. In this study, we show that PPAR-α is essential for antimycobacterial responses via activation of transcription factor EB (TFEB) transcription and inhibition of lipid body formation. PPAR-α deficiency resulted in an increased bacterial load and exaggerated inflammatory responses during mycobacterial infection. PPAR-α agonists promoted autophagy, lysosomal biogenesis, phagosomal maturation, and antimicrobial defense against Mycobacterium tuberculosis or M. bovis bacillus Calmette-Guérin. PPAR-α agonists regulated multiple genes involved in autophagy and lysosomal biogenesis, including Lamp2, Rab7, and Tfeb in bone marrow-derived macrophages. Silencing of TFEB reduced phagosomal maturation and antimicrobial responses, but increased macrophage inflammatory responses during mycobacterial infection. Moreover, PPAR-α activation promoted lipid catabolism and fatty acid ß-oxidation in macrophages during mycobacterial infection. Taken together, our data indicate that PPAR-α mediates antimicrobial responses to mycobacterial infection by inducing TFEB and lipid catabolism.

Thiostrepton: A Novel Therapeutic Drug Candidate for Mycobacterium abscessus Infection.

Mycobacterium abscessus is a rapid-growing, multidrug-resistant, non-tuberculous mycobacterial species responsible for a variety of human infections, such as cutaneous and pulmonary infections. M. abscessus infections are very difficult to eradicate due to the natural and acquired multidrug resistance profiles of M. abscessus. Thus, there is an urgent need for the development of effective drugs or regimens against M. abscessus infections. Here, we report the activity of a US Food and Drug Administration approved drug, thiostrepton, against M. abscessus. We found that thiostrepton significantly inhibited the growth of M. abscessus wild-type strains, subspecies, clinical isolates, and drug-resistant mutants in vitro and in macrophages. In addition, treatment of macrophages with thiostrepton significantly decreased proinflammatory cytokine production in a dose-dependent manner, suggesting an inhibitory effect of thiostrepton on inflammation induced during M. abscessus infection. We further showed that thiostrepton exhibits antimicrobial effects in vivo using a zebrafish model of M. abscessus infection.

Conformation-Enabled Total Syntheses of Ohmyungsamycins†A and B and Structural Revision of Ohmyungsamycin†B.

The first total syntheses of the bioactive cyclodepsipeptides ohmyungsamycin A and B are described. Key features of our synthesis include the concise preparation of a linear cyclization precursor that consists of N-methyl amides and non-proteinogenic amino acids, and its macrolactamization from a bent conformation. The proposed structure of ohmyungsamycin B was revised based on its synthesis. The cyclic core of the ohmyungsamycins was shown to be responsible for the excellent antituberculosis activity, and ohmyungsamycin variants with truncated chains were evaluated for their biological activity.

Efflux Attenuates the Antibacterial Activity of Q203 in Mycobacterium tuberculosis.

New and improved treatments for tuberculosis (TB) are urgently needed. Recently, it has been demonstrated that verapamil, an efflux inhibitor, can reduce bacterial drug tolerance caused by efflux pump activity when administered in combination with available antituberculosis agents. The aim of this study was to evaluate the effectiveness of verapamil in combination with the antituberculosis drug candidate Q203, which has recently been developed and is currently under clinical trials as a potential antituberculosis agent. We evaluated changes in Q203 activity in the presence and absence of verapamil in vitro using the resazurin microplate assay and ex vivo using a microscopy-based phenotypic assay for the quantification of intracellular replicating mycobacteria. Verapamil increased the potency of Q203 against Mycobacterium tuberculosis both in vitro and ex vivo, indicating that efflux pumps are associated with the activity of Q203. Other efflux pump inhibitors also displayed an increase in Q203 potency, strengthening this hypothesis. Therefore, the combination of verapamil and Q203 may be a promising combinatorial strategy for anti-TB treatment to accelerate the elimination of M. tuberculosis.

Activity of LCB01-0371, a Novel Oxazolidinone, against Mycobacterium abscessus.

Mycobacterium abscessus is a highly pathogenic drug-resistant rapidly growing mycobacterium. In this study, we evaluated the in vitro, intracellular, and in vivo activities of LCB01-0371, a novel and safe oxazolidinone derivative, for the treatment of M. abscessus infection and compared its resistance to that of other oxazolidinone drugs. LCB01-0371 was effective against several M. abscessus strains in vitro and in a macrophage model of infection. In the murine model, a similar efficacy to linezolid was achieved, especially in the lungs. We induced laboratory-generated resistance to LCB01-0371; sequencing analysis revealed mutations in rplC of T424C and G419A and a nucleotide insertion at the 503 position. Furthermore, LCB01-0371 inhibited the growth of amikacin-, cefoxitin-, and clarithromycin-resistant strains. Collectively, our data indicate that LCB01-0371 might represent a promising new class of oxazolidinones with improved safety, which may replace linezolid for the treatment of M. abscessus.

Isolation and Characterization of a Hybrid Respiratory Supercomplex Consisting of Mycobacterium tuberculosis Cytochrome bcc and Mycobacterium smegmatis Cytochrome aa3.

Recently, energy production pathways have been shown to be viable antitubercular drug targets to combat multidrug-resistant tuberculosis and eliminate pathogen in the dormant state. One family of drugs currently under development, the imidazo[1,2-a]pyridine derivatives, is believed to target the pathogen's homolog of the mitochondrial bc1 complex. This complex, denoted cytochrome bcc, is highly divergent from mitochondrial Complex III both in subunit structure and inhibitor sensitivity, making it a good target for drug development. There is no soluble cytochrome c in mycobacteria to transport electrons from the bcc complex to cytochrome oxidase. Instead, the bcc complex exists in a "supercomplex" with a cytochrome aa3-type cytochrome oxidase, presumably allowing direct electron transfer. We describe here purification and initial characterization of the mycobacterial cytochrome bcc-aa3 supercomplex using a strain of M. smegmatis that has been engineered to express the M. tuberculosis cytochrome bcc. The resulting hybrid supercomplex is stable during extraction and purification in the presence of dodecyl maltoside detergent. It is hoped that this purification procedure will potentiate functional studies of the complex as well as crystallographic studies of drug binding and provide structural insight into a third class of the bc complex superfamily.

para-Aminosalicylic acid is a prodrug targeting dihydrofolate reductase in Mycobacterium tuberculosis.

para-Aminosalicylic acid (PAS) is one of the antimycobacterial drugs currently used for multidrug-resistant tuberculosis. Although it has been in clinical use for over 60 years, its mechanism(s) of action remains elusive. Here we report that PAS is a prodrug targeting dihydrofolate reductase (DHFR) through an unusual and novel mechanism of action. We provide evidences that PAS is incorporated into the folate pathway by dihydropteroate synthase (DHPS) and dihydrofolate synthase (DHFS) to generate a hydroxyl dihydrofolate antimetabolite, which in turn inhibits DHFR enzymatic activity. Interestingly, PAS is recognized by DHPS as efficiently as its natural substrate para-amino benzoic acid. Chemical inhibition of DHPS or mutation in DHFS prevents the formation of the antimetabolite, thereby conferring resistance to PAS. In addition, we identified a bifunctional enzyme (riboflavin biosynthesis protein (RibD)), a putative functional analog of DHFR in a knock-out strain. This finding is further supported by the identification of PAS-resistant clinical isolates encoding a RibD overexpression mutation displaying cross-resistance to genuine DHFR inhibitors. Our findings reveal that a metabolite of PAS inhibits DHFR in the folate pathway. RibD was shown to act as a functional analog of DHFR, and as for DHFS, both were shown to be associated in PAS resistance in laboratory strains and clinical isolates.

Novel drug combination for Mycobacterium abscessus disease therapy identified in a Drosophila infection model.

OBJECTIVES: Mycobacterium abscessus is known to be the most drug-resistant Mycobacterium and accounts for ∼80% of pulmonary infections caused by rapidly growing mycobacteria. This study reports a new Drosophila melanogaster-M. abscessus infection model that can be used as an in vivo efficacy model for anti-M. abscessus drug potency assessment. METHODS: D. melanogaster were challenged with M. abscessus, and infected flies were fed with a fly medium containing tigecycline, clarithromycin, linezolid, clofazimine, moxifloxacin, amikacin, cefoxitin, dinitrobenzamide or metronidazole at different concentrations (0, 100 and 500 mg/L). The survival rates of infected flies were plotted and bacterial colonization/dissemination in fly bodies was monitored by cfu determination and green fluorescent protein epifluorescence. RESULTS: The D. melanogaster-M. abscessus model enabled an assessment of the effectiveness of antibiotic treatment. Tigecycline was the best drug for extending the lifespan of M. abscessus-infected Drosophila, followed by clarithromycin and linezolid. Several different combinations of tigecycline, linezolid and clarithromycin were tested to determine the best combination. Tigecycline (25 mg/L) plus linezolid (500 mg/L) was the best drug combination and its efficacy was superior to conventional regimens, not only in prolonging infected fly survival but also against M. abscessus colonization and dissemination. CONCLUSIONS: This D. melanogaster-M. abscessus infection/curing methodology may be useful for the rapid evaluation of potential drug candidates. In addition, new combinations using tigecycline and linezolid should be considered as possible next-generation combination therapies to be assessed in higher organisms.

Exploring antibiotic resistance mechanisms in Mycobacterium abscessus for enhanced therapeutic approaches.

Mycobacterium abscessus, a leading cause of severe lung infections in immunocompromised individuals, poses significant challenges for current therapeutic strategies due to resistance mechanisms. Therefore, understanding the intrinsic and acquired antibiotic resistance of M. abscessus is crucial for effective treatment. This review highlights the mechanisms employed by M. abscessus to sustain antibiotic resistance, encompassing not only conventional drugs but also newly discovered drug candidates. This comprehensive analysis aims to identify novel entities capable of overcoming the notorious resistance exhibited by M. abscessus, providing insights for the development of more effective therapeutic interventions.