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.

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.

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.

CRISPR Interference-Based Inhibition of MAB_0055c Expression Alters Drug Sensitivity in Mycobacterium abscessus.

There is an unmet medical need for effective treatments against Mycobacterium abscessus infections. Although advanced molecular genetic tools to validate drug targets and resistance of M. abscessus exist, the practical design and construction of plasmids are relatively laborious and time-consuming. Thus, for this purpose, we used CRISPR interference (CRISPRi) combined with catalytically deactivated Cas9 to inhibit the gene expression of a predicted LysR-type transcriptional regulator gene, MAB_0055c, in M. abscessus and evaluated its contribution to the development of drug resistance. Our results showed that silencing the MAB_0055c gene lead to increased rifamycin susceptibility depending on the hydroquinone moiety. These results demonstrate that CRISPRi is an excellent approach for studying drug resistance in M. abscessus. IMPORTANCE In this study, we utilized CRISPR interference (CRISPRi) to specifically target the MAB_0055c gene in M. abscessus, a bacterium that causes difficult-to-treat infections. The study found that silencing the gene lead to increased rifabutin and rifalazil susceptibility. This study is the first to establish a link between the predicted LysR-type transcriptional regulator gene and antibiotic resistance in mycobacteria. These findings underscore the potential of using CRISPRi as a tool for elucidating resistance mechanisms, essential drug targets, and drug mechanisms of action, which could pave the way for more effective treatments for M. abscessus infections. The results of this study could have important implications for the development of new therapeutic options for this challenging-to-treat bacterial infection.

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.

Dimethyl itaconate is effective in host-directed antimicrobial responses against mycobacterial infections through multifaceted innate immune pathways.

BACKGROUND: Itaconate, a crucial immunometabolite, plays a critical role in linking immune and metabolic functions to influence host defense and inflammation. Due to its polar structure, the esterified cell-permeable derivatives of itaconate are being developed to provide therapeutic opportunities in infectious and inflammatory diseases. Yet, it remains largely uncharacterized whether itaconate derivatives have potentials in promoting host-directed therapeutics (HDT) against mycobacterial infections. Here, we report dimethyl itaconate (DMI) as the promising candidate for HDT against both Mycobacterium tuberculosis (Mtb) and nontuberculous mycobacteria by orchestrating multiple innate immune programs. RESULTS: DMI per se has low bactericidal activity against Mtb, M. bovis Bacillus Calmette-Guérin (BCG), and M. avium (Mav). However, DMI robustly activated intracellular elimination of multiple mycobacterial strains (Mtb, BCG, Mav, and even to multidrug-resistant Mtb) in macrophages and in vivo. DMI significantly suppressed the production of interleukin-6 and -10, whereas it enhanced autophagy and phagosomal maturation, during Mtb infection. DMI-mediated autophagy partly contributed to antimicrobial host defenses in macrophages. Moreover, DMI significantly downregulated the activation of signal transducer and activator of transcription 3 signaling during infection with Mtb, BCG, and Mav. CONCLUSION: Together, DMI has potent anti-mycobacterial activities in macrophages and in vivo through promoting multifaceted ways for innate host defenses. DMI may bring light to new candidate for HDT against Mtb and nontuberculous mycobacteria, both of which infections are often intractable with antibiotic resistance.

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.

Ohmyungsamycin promotes M1-like inflammatory responses to enhance host defence against Mycobacteroides abscessus infections.

Ohmyungsamycin A (OMS) is a newly identified cyclic peptide that exerts antimicrobial effects against Mycobacterium tuberculosis. However, its role in nontuberculous mycobacteria (NTMs) infections has not been clarified. Mycobacteroides abscessus (Mabc) is a rapidly growing NTM that has emerged as a human pathogen in both immunocompetent and immunosuppressed individuals. In this study, we demonstrated that OMS had significant antimicrobial effects against Mabc infection in both immunocompetent and immunodeficient mice, and in macrophages. OMS treatment amplified Mabc-induced expression of M1-related proinflammatory cytokines and inducible nitric oxide synthase, and significantly downregulated arginase-1 expression in murine macrophages. In addition, OMS augmented Mabc-mediated production of mitochondrial reactive oxygen species (mtROS), which promoted M1-like proinflammatory responses in Mabc-infected macrophages. OMS-induced production of mtROS and nitric oxide was critical for OMS-mediated antimicrobial responses during Mabc infections. Notably, the combination of OMS and rifabutin had a synergistic effect on the antimicrobial responses against Mabc infections in vitro, in murine macrophages, and in zebrafish models in vivo. Collectively, these data strongly suggest that OMS may be an effective M1-like adjunctive therapeutic against Mabc infections, either alone or in combination with antibiotics.

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.

PE_PGRS38 Interaction With HAUSP Downregulates Antimycobacterial Host Defense via TRAF6.

Mycobacterium tuberculosis (Mtb) is the causative pathogen of tuberculosis (TB), which manipulates the host immunity to ensure survival and colonization in the host. Mtb possess a unique family of proteins, named PE_PGRS, associated with Mtb pathogenesis. Thus, elucidation of the functions of PE_PGRS proteins is necessary to understand TB pathogenesis. Here, we investigated the role of PE_PGRS38 binding to herpesvirus-associated ubiquitin-specific protease (HAUSP, USP7) in regulating the activity of various substrate proteins by modulating their state of ubiquitination. We constructed the recombinant PE_PGRS38 expressed in M. smegmatis (Ms_PE_PGRS38) to investigate the role of PE_PGRS38. We found that Ms_PE_PGRS38 regulated the cytokine levels in murine bone marrow-derived macrophages by inhibiting the deubiquitination of tumor necrosis factor receptor-associated factor (TRAF) 6 by HAUSP. Furthermore, the PE domain in PE_PGRS38 was identified as essential for mediating TRAF6 deubiquitination. Ms_PE_PGRS38 increased the intracellular burden of bacteria by manipulating cytokine levels in vitro and in vivo. Overall, we revealed that the interplay between HAUSP and PE_PGRS38 regulated the inflammatory response to increase the survival of mycobacteria.

A combination screening to identify enhancers of para-aminosalicylic acid against Mycobacterium tuberculosis.

Para-aminosalicylic acid (PAS) is an antibiotic that was largely used for the multi-therapy of tuberculosis in the twentieth century. To try to overcome the inconvenience of its low efficacy and poor tolerance, we searched for novel chemical entities able to synergize with PAS using a combination screening against growing axenic Mycobacterium tuberculosis. The screening was performed at a sub-inhibitory concentration of PAS on a library of about 100,000 small molecules. Selected hit compounds were analyzed by dose-response and further probed with an intracellular macrophage assay. Scaffolds with potential additive effect with PAS are reported, opening interesting prospects for mechanism of action studies. We also report here evidence of a yet unknown bio-activation mechanism, involving activation of pyrido[1,2-a]pyrimidin-4-one (PP) derivatives through the Rv3087 protein.

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).

Omadacycline Potentiates Clarithromycin Activity Against Mycobacterium abscessus.

Mycobacterium abscessus is a difficult respiratory pathogen to treat, when compared to other nontuberculus mycobacteria (NTM), due to its drug resistance. In this study, we aimed to find a new clarithromycin partner that potentiated strong, positive, synergy against M. abscessus among current anti-M. abscessus drugs, including omadacycline, amikacin, rifabutin, bedaquiline, and cefoxitine. First, we determined the minimum inhibitory concentrations required of all the drugs tested for M. abscessus subsp. abscessus CIP104536T treatment using a resazurin microplate assay. Next, the best synergistic partner for clarithromycin against M. abscessus was determined using an in vitro checkerboard combination assay. Among the drug combinations evaluated, omadacycline showed the best synergistic effect with clarithromycin, with a fractional inhibitory concentration index of 0.4. This positive effect was also observed against M. abscessus clinical isolates and anti-M. abscessus drug resistant strains. Lastly, this combination was further validated using a M. abscessus infected zebrafish model. In this model, the clarithromycin-omadacyline regimen was found to inhibit the dissemination of M. abscessus, and it significantly extended the lifespan of the M. abscessus infected zebrafish. In summation, the synergy between two anti-M. abscessus compounds, clarithromycin and omadacycline, provides an attractive foundation for a new M. abscessus treatment regimen.

Current Molecular Therapeutic Agents and Drug Candidates for Mycobacterium abscessus.

Mycobacterium abscessus has been recognised as a dreadful respiratory pathogen among the non-tuberculous mycobacteria (NTM) because of misdiagnosis, prolonged therapy with poor treatment outcomes and a high cost. This pathogen also shows extremely high antimicrobial resistance against current antibiotics, including the anti-tuberculosis agents. Therefore, current chemotherapies require a long curative period and the clinical outcomes are not satisfactory. Thus, there is an urgent need for discovering and developing novel, more effective anti-M. abscessus drugs. In this review, we sum the effectiveness of the current anti-M. abscessus drugs and drug candidates. Furthermore, we describe the shortcomings and difficulties associated with M. abscessus drug discovery and development.

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.

Dumulmycin, an Antitubercular Bicyclic Macrolide from a Riverine Sediment-Derived Streptomyces sp.

Dumulmycin (1) was isolated from Streptomyces sp. DM28, a bacterial strain from a riverine sediment sample. The structure of 1 was elucidated as a bicyclic macrolide possessing 19-membered and 5-membered rings by spectroscopic analysis. The stereochemistry of 1 was determined by J-based configuration analysis, ROESY NMR data, DP4 calculations, and the modified Mosher's method. Genetic analysis identified a trans-acyltransferase polyketide biosynthetic gene cluster for 1. Dumulmycin exhibited in vitro antitubercular activity (MIC50 = 27.1 µM).

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.

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.

Sirtuin 3 is essential for host defense against Mycobacterium abscessus infection through regulation of mitochondrial homeostasis.

The global incidence of Mycobacterium abscessus (Mabc), a rapidly growing nontuberculous mycobacterial strain that causes treatment-refractory pulmonary diseases, is increasing. Despite this, the host factors that allow for protection against infection are largely unknown. In this study, we found that sirtuin 3 (SIRT3), a mitochondrial protein deacetylase, plays a critical role in host defense against Mabc infection. Mabc decreased SIRT3 and upregulated mitochondrial oxidative stress in macrophages. SIRT3 deficiency led to increased bacterial loads, histopathological, and mitochondrial damage, and pathological inflammation during Mabc infection. Administration of scavengers of mitochondrial reactive oxygen species significantly decreased the in vivo Mabc burden and excessive inflammation, and induced SIRT3 expression in infected lungs. Notably, SIRT3 agonist (resveratrol) significantly decreased Mabc growth and attenuated inflammation in mice and zebrafishes, indicating the key role for SIRT3 in metazoan host defense. Collectively, these data strongly suggest that SIRT3 is a host-directed therapeutic target against Mabc infection by controlling mitochondrial homeostasis.

Structural Revision of Lydiamycin A by Reinvestigation of the Stereochemistry.

Lydiamycin A (1) is a previously reported piperazic acid-bearing cyclic peptide from Streptomyces. The absolute configuration of lydiamycin A has not been fully determined despite the fact that multiple total syntheses have been reported. The absolute configuration of 1 was reinvestigated by the advanced Marfey's method, chemical derivatization, and quantum-mechanics-based computational analysis, eventually resulting in a structural revision and establishment of the complete configuration. Lydiamycin A displayed weak antituberculosis activity in vitro.