Streptomyces extracellular vesicles are a broad and permissive antimicrobial packaging and delivery system.

Streptomyces are the primary source of bioactive specialized metabolites used in research and medicine, including many antimicrobials. These are presumed to be secreted and function as freely soluble compounds. However, increasing evidence suggests that extracellular vesicles are an alternative secretion system. We assessed environmental and lab-adapted Streptomyces (sporulating filamentous actinomycetes) and found frequent production of antimicrobial vesicles. The molecular cargo included actinomycins, anthracyclines, candicidin, and actinorhodin, reflecting both diverse chemical properties and diverse antibacterial and antifungal activity. The levels of packaged antimicrobials correlated with the level of inhibitory activity of the vesicles, and a strain knocked out for the production of anthracyclines produced vesicles that lacked antimicrobial activity. We demonstrated that antimicrobial containing vesicles achieve direct delivery of the cargo to other microbes. Notably, this delivery via membrane fusion occurred to a broad range of microbes, including pathogenic bacteria and yeast. Vesicle encapsulation offers a broad and permissive packaging and delivery system for antimicrobial specialized metabolites, with important implications for ecology and translation.IMPORTANCEExtracellular vesicle encapsulation changes our picture of how antimicrobial metabolites function in the environment and provides an alternative translational approach for the delivery of antimicrobials. We find many Streptomyces strains are capable of releasing antimicrobial vesicles, and at least four distinct classes of compounds can be packaged, suggesting this is widespread in nature. This is a striking departure from the primary paradigm of the secretion and action of specialized metabolites as soluble compounds. Importantly, the vesicles deliver antimicrobial metabolites directly to other microbes via membrane fusion, including pathogenic bacteria and yeast. This suggests future applications in which lipid-encapsulated natural product antibiotics and antifungals could be used to solve some of the most pressing problems in drug resistance.

Targeting fungal membrane homeostasis with imidazopyrazoindoles impairs azole resistance and biofilm formation.

Fungal infections cause more than 1.5 million deaths annually. With an increase in immune-deficient susceptible populations and the emergence of antifungal drug resistance, there is an urgent need for novel strategies to combat these life-threatening infections. Here, we use a combinatorial screening approach to identify an imidazopyrazoindole, NPD827, that synergizes with fluconazole against azole-sensitive and -resistant isolates of Candida albicans. NPD827 interacts with sterols, resulting in profound effects on fungal membrane homeostasis and induction of membrane-associated stress responses. The compound impairs virulence in a Caenorhabditis elegans model of candidiasis, blocks C. albicans filamentation in vitro, and prevents biofilm formation in a rat model of catheter infection by C. albicans. Collectively, this work identifies an imidazopyrazoindole scaffold with a non-protein-targeted mode of action that re-sensitizes the leading human fungal pathogen, C. albicans, to azole antifungals.

High-Throughput Chemical Screen Identifies a 2,5-Disubstituted Pyridine as an Inhibitor of Candida albicans Erg11.

Fungal infections contribute to over 1.5 million deaths annually, with Candida albicans representing one of the most concerning human fungal pathogens. While normally commensal in nature, compromise of host immunity can result in C. albicans disseminating into the human bloodstream, causing infections with mortality rates of up to 40%. A contributing factor to this high mortality rate is the limited arsenal of antifungals approved to treat systemic infections. The most widely used antifungal class, the azoles, inhibits ergosterol biosynthesis by targeting Erg11. The rise of drug resistance among C. albicans clinical isolates, particularly against the azoles, has escalated the need to explore novel antifungal strategies. To address this challenge, we screened a 9,600-compound subset of the University of Tokyo Core Chemical Library to identify molecules with novel antifungal activity against C. albicans. The most potent hit molecule was CpdLC-6888, a 2,5-disubstituted pyridine compound, which inhibited growth of C. albicans and closely-related species. Chemical-genetic, biochemical, and modeling analyses suggest that CpdLC-6888 inhibits Erg11 in a manner similar to the azoles despite lacking the canonical five-membered nitrogen-containing azole ring. This work characterizes the antifungal activity of a 2,5-disubstituted pyridine against C. albicans, supporting the mining of existing chemical collections to identify compounds with novel antifungal activity. IMPORTANCE Pathogenic fungi represent a serious but underacknowledged threat to human health. The treatment and management of these infections relies heavily on the use of azole antifungals, a class of molecules that contain a five-membered nitrogen-containing ring and inhibit the biosynthesis of the key membrane sterol ergosterol. By employing a high-throughput chemical screen, we identified a 2,5-disubstituted pyridine, termed CpdLC-6888, as possessing antifungal activity against the prominent human fungal pathogen Candida albicans. Upon further investigation, we determined this molecule exhibits azole-like activity despite being structurally divergent. Specifically, transcriptional repression of the azole target gene ERG11 resulted in hypersensitivity to CpdLC-6888, and treatment of C. albicans with this molecule blocked the production of the key membrane sterol ergosterol. Therefore, this work describes a chemical scaffold with novel antifungal activity against a prevalent and threatening fungal pathogen affecting human health, expanding the repertoire of compounds that can inhibit this useful antifungal drug target.

Cytosolic and Mitochondrial Hsp90 in Cytokinesis, Mitochondrial DNA Replication, and Drug Action in Trypanosoma brucei.

Trypanosoma brucei subspecies cause African sleeping sickness in humans, an infection that is commonly fatal if not treated, and available therapies are limited. Previous studies have shown that heat shock protein 90 (Hsp90) inhibitors have potent and vivid activity against bloodstream-form trypanosomes. Hsp90s are phylogenetically conserved and essential catalysts that function at the crux of cell biology, where they ensure the proper folding of proteins and their assembly into multicomponent complexes. To assess the specificity of Hsp90 inhibitors and further define the role of Hsp90s in African trypanosomes, we used RNA interference (RNAi) to knock down cytosolic and mitochondrial Hsp90s (HSP83 and HSP84, respectively). Loss of either protein led to cell death, but the phenotypes were distinctly different. Depletion of cytosolic HSP83 closely mimicked the consequences of chemically depleting Hsp90 activity with inhibitor 17-AAG. In these cells, cytokinesis was severely disrupted, and segregation of the kinetoplast (the massive mitochondrial DNA structure unique to this family of eukaryotic pathogens) was impaired, leading to cells with abnormal kinetoplast DNA (kDNA) structures. Quite differently, knockdown of mitochondrial HSP84 did not impair cytokinesis but halted the initiation of new kDNA synthesis, generating cells without kDNA. These findings highlight the central role of Hsp90s in chaperoning cell cycle regulators in trypanosomes, reveal their unique function in kinetoplast replication, and reinforce their specificity and value as drug targets.

Biology and applications of co-produced, synergistic antimicrobials from environmental bacteria.

Environmental bacteria, such as Streptomyces spp., produce specialized metabolites that are potent antibiotics and therapeutics. Selected specialized antimicrobials are co-produced and function together synergistically. Co-produced antimicrobials comprise multiple chemical classes and are produced by a wide variety of bacteria in different environmental niches, suggesting that their combined functions are ecologically important. Here, we highlight the exquisite mechanisms that underlie the simultaneous production and functional synergy of 16 sets of co-produced antimicrobials. To date, antibiotic and antifungal discovery has focused mainly on single molecules, but we propose that methods to target co-produced antimicrobials could widen the scope and applications of discovery programs.

Predicting antimicrobial mechanism-of-action from transcriptomes: A generalizable explainable artificial intelligence approach.

To better combat the expansion of antibiotic resistance in pathogens, new compounds, particularly those with novel mechanisms-of-action [MOA], represent a major research priority in biomedical science. However, rediscovery of known antibiotics demonstrates a need for approaches that accurately identify potential novelty with higher throughput and reduced labor. Here we describe an explainable artificial intelligence classification methodology that emphasizes prediction performance and human interpretability by using a Hierarchical Ensemble of Classifiers model optimized with a novel feature selection algorithm called Clairvoyance; collectively referred to as a CoHEC model. We evaluated our methods using whole transcriptome responses from Escherichia coli challenged with 41 known antibiotics and 9 crude extracts while depositing 122 transcriptomes unique to this study. Our CoHEC model can properly predict the primary MOA of previously unobserved compounds in both purified forms and crude extracts at an accuracy above 99%, while also correctly identifying darobactin, a newly discovered antibiotic, as having a novel MOA. In addition, we deploy our methods on a recent E. coli transcriptomics dataset from a different strain and a Mycobacterium smegmatis metabolomics timeseries dataset showcasing exceptionally high performance; improving upon the performance metrics of the original publications. We not only provide insight into the biological interpretation of our model but also that the concept of MOA is a non-discrete heuristic with diverse effects for different compounds within the same MOA, suggesting substantial antibiotic diversity awaiting discovery within existing MOA.

Pulse Dosing of Antibiotic Enhances Killing of a Staphylococcus aureus Biofilm.

Biofilms are highly tolerant to antibiotics and underlie the recalcitrance of many chronic infections. We demonstrate that mature Staphylococcus aureus biofilms can be substantially sensitized to the treatment by pulse dosing of an antibiotic - in this case, oxacillin. Pulse (periodic) dosing was compared to continuous application of antibiotic and was studied in a novel in vitro flow system which allowed for robust biofilm growth and tractable pharmacokinetics of dosing regimens. Our results highlight that a subpopulation of the biofilm survives antibiotic without becoming resistant, a population we refer to as persister bacteria. When oxacillin was continuously present the persister level did not decline, but, importantly, providing correctly timed periodic breaks decreased the surviving population. We found that the length of the periodic break impacted efficacy, and there was an optimal length that sensitized the biofilm to repeat treatment without allowing resistance expansion. Periodic dosing provides a potential simple solution to a complicated problem.

Author Correction: A new antibiotic selectively kills Gram-negative pathogens.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Mechanism-of-Action Classification of Antibiotics by Global Transcriptome Profiling.

Antimicrobial resistance (AMR) is an ever-growing public health problem worldwide. The low rate of antibiotic discovery coupled with the rapid spread of drug-resistant bacterial pathogens is causing a global health crisis. To facilitate the drug discovery processes, we present a large-scale study of reference antibiotic challenge bacterial transcriptome profiles, which included 37 antibiotics across 6 mechanisms of actions (MOAs) and provide an economical approach to aid in antimicrobial dereplication in the discovery process. We demonstrate that classical MOAs can be sorted based upon the magnitude of gene expression profiles despite some overlap in the secondary effects of antibiotic exposures across MOAs. Additionally, using gene subsets, we were able to subdivide broad MOA classes into subMOAs. Furthermore, we provide a biomarker gene set that can be used to classify most antimicrobial challenges according to their canonical MOA. We also demonstrate the ability of this rapid MOA diagnostic tool to predict and classify the expression profiles of pure compounds and crude extracts to their expression profile-associated MOA class.

A new antibiotic selectively kills Gram-negative pathogens.

The current need for novel antibiotics is especially acute for drug-resistant Gram-negative pathogens1,2. These microorganisms have a highly restrictive permeability barrier, which limits the penetration of most compounds3,4. As a result, the last class of antibiotics that acted against Gram-negative bacteria was developed in the 1960s2. We reason that useful compounds can be found in bacteria that share similar requirements for antibiotics with humans, and focus on Photorhabdus symbionts of entomopathogenic nematode microbiomes. Here we report a new antibiotic that we name darobactin, which was obtained using a screen of Photorhabdus isolates. Darobactin is coded by a silent operon with little production under laboratory conditions, and is ribosomally synthesized. Darobactin has an unusual structure with two fused rings that form post-translationally. The compound is active against important Gram-negative pathogens both in vitro and in animal models of infection. Mutants that are resistant to darobactin map to BamA, an essential chaperone and translocator that folds outer membrane proteins. Our study suggests that bacterial symbionts of animals contain antibiotics that are particularly suitable for development into therapeutics.

Gram-scale total synthesis of teixobactin promoting binding mode study and discovery of more potent antibiotics.

Teixobactin represents a new class of antibiotics with novel structure and excellent activity against Gram-positive pathogens and Mycobacterium tuberculosis. Herein, we report a one-pot reaction to conveniently construct the key building block L-allo-Enduracidine in 30-gram scale in just one hour and a convergent strategy (3 + 2 + 6) to accomplish a gram-scale total synthesis of teixobactin. Several analogs are described, with 20 and 26 identified as the most efficacious analogs with 3~8-fold and 2~4-fold greater potency against vancomycin resistant Enterococcus faecalis and methicillin-resistant Staphylococcus aureus respectively in comparison with teixobactin. In addition, they show high efficiency in Streptococcus pneumoniae septicemia mouse model and neutropenic mouse thigh infection model using methicillin-resistant Staphylococcus aureus. We also propose that the antiparallel ß-sheet of teixobactin is important for its bioactivity and an antiparallel dimer of teixobactin is the minimal binding unit for lipid II via key amino acids variations and molecular docking.

Optimal kinetic exposures for classic and candidate antitrypanosomals.

OBJECTIVES: Efficacy is determined not only by size, but also by shape, of drug exposure. Here the critical importance of the temporal pattern of drug concentrations (pharmacokinetic profile) is examined for antitrypanosomals in vitro. METHODS: An in vitro hollow-fibre cartridge system was used to study contrasting drug profiles with four clinically used agents and two experimental candidates against the deadly parasite Trypanosoma brucei. Artificial kinetics were employed intentionally to favour either high peak concentration or sustained duration of drug. RESULTS: Changing the shape of drug exposure significantly impacted drug efficacy. Suramin, melarsoprol and pentamidine were concentration-driven and therefore more efficacious when applied as short-lived high peaks. In contrast, difluoromethylornithine (DFMO) was time-driven, and therefore maximally effective as a constant infusion. Kinetic preference was robust over a wide range of drug exposures. Promising clinical candidates SCYX-7158 (acoziborole) and fexinidazole (parent and sulfone) were concentration-driven, suggesting optimal clinical regimens would involve relatively high but intermittent dosing. CONCLUSIONS: Antitrypanosomals have an intrinsic pharmacokinetic driver for optimal efficacy, with important implications for clinical management and future candidate development.

Model System Identifies Kinetic Driver of Hsp90 Inhibitor Activity against African Trypanosomes and Plasmodium falciparum.

Hsp90 inhibitors, well studied in the laboratory and clinic for antitumor indications, have promising activity against protozoan pathogens, including Trypanosoma brucei which causes African sleeping sickness, and the malaria parasite, Plasmodium falciparum To progress these experimental drugs toward clinical use, we adapted an in vitro dynamic hollow-fiber system and deployed artificial pharmacokinetics to discover the driver of their activity: either concentration or time. The activities of compounds from three major classes of Hsp90 inhibitors in development were evaluated against trypanosomes. In all circumstances, the activities of the tested Hsp90 inhibitors were concentration driven. By optimally deploying the drug to match its kinetic driver, the efficacy of a given dose was improved up to 5-fold, and maximal efficacy was achieved with a significantly lower drug exposure. The superiority of concentration-driven regimens was evident in vitro over several logs of drug exposure and was predictive of efficacy in a mouse model of African trypanosomiasis. In studies with P. falciparum, antimalarial activity was similarly concentration driven. This experimental strategy offers an expedient and versatile translational tool to assess the impact of pharmacokinetics on antiprotozoal activity. Knowing kinetic governance early in drug development provides an additional metric for judging lead compounds and allows the incisive design of animal efficacy studies.

Developing Equipotent Teixobactin Analogues against Drug-Resistant Bacteria and Discovering a Hydrophobic Interaction between Lipid II and Teixobactin.

Teixobactin, targeting lipid II, represents a new class of antibiotics with novel structures and has excellent activity against Gram-positive pathogens. We developed a new convergent method to synthesize a series of teixobactin analogues and explored structure-activity relationships. We obtained equipotent and simplified teixobactin analogues, replacing the l- allo-enduracididine with lysine, substituting oxygen to nitrogen on threonine, and adding a phenyl group on the d-phenylalanine. On the basis of the antibacterial activities that resulted from corresponding modifications of the d-phenylalanine, we propose a hydrophobic interaction between lipid II and the N-terminal of teixobactin analogues, which we map out with our analogue 35. Finally, a representative analogue from our series showed high efficiency in a mouse model of Streptococcus pneumoniae septicemia.

Creative Arts Interventions to Address Depression in Older Adults: A Systematic Review of Outcomes, Processes, and Mechanisms.

Depression experienced by older adults is proving an increasing global health burden, with rates generally 7% and as high as 27% in the USA. This is likely to significantly increase in coming years as the number and proportion of older adults in the population rises all around the world. Therefore, it is imperative that the effectiveness of approaches to the prevention and treatment of depression are understood. Creative arts interventions, including art, dance movement, drama, and music modalities, are utilized internationally to target depression and depressive symptoms in older adults. This includes interventions led by trained arts therapists as well as other health and arts professionals. However, to date there has not been a systematic review that reports effects and examines the processes (why) and mechanisms (how) of creative arts interventions are used to address depression in this older age group. This systematic review of studies on creative arts interventions for older adults experiencing depression examined: outcomes of four creative arts modalities (art, dance movement, drama, and music); with particular attention paid to processes documented as contributing to change in each modality; and mechanisms considered to result from these processes. Our analysis of 75 articles (17 art, 13 dance, 4 drama, and 41 music) indicates mostly significant quantitative or positive qualitative findings, particularly for interventions led by creative arts therapists. Mechanisms of change gleaned from the studies that were common across modalities include physical (e.g., increased muscle strength; neurochemical effects, such as endorphin release), intra-personal (e.g., enhanced self-concept, strengthened agency and mastery; processing and communication of emotions), cultural (e.g., creative expression, aesthetic pleasure), cognitive (e.g., stimulation of memory), and social (e.g., increased social skills and connection), that were all considered to contribute to reduced depression and symptoms. Recommendations for future research includes stronger focus on testing of processes and mechanisms.

Potent antitrypanosomal activities of heat shock protein 90 inhibitors in vitro and in vivo.

African sleeping sickness, caused by the protozoan parasite Trypanosoma brucei, is universally fatal if untreated, and current drugs are limited by severe toxicities and difficult administration. New antitrypanosomals are greatly needed. Heat shock protein 90 (Hsp90) is a conserved and ubiquitously expressed molecular chaperone essential for stress responses and cellular signaling. We investigated Hsp90 inhibitors for their antitrypanosomal activity. Geldanamycin and radicicol had nanomolar potency in vitro against bloodstream-form T. brucei; novobiocin had micromolar activity. In structure-activity studies of geldanamycin analogs, 17-AAG and 17-DMAG were most selective against T. brucei as compared to mammalian cells. 17-AAG treatment sensitized trypanosomes to heat shock and caused severe morphological abnormalities and cell cycle disruption. Both oral and parenteral 17-DMAG cured mice of a normally lethal infection of T. brucei. These promising results support the use of inhibitors to study Hsp90 function in trypanosomes and to expand current clinical development of Hsp90 inhibitors to include T. brucei.

Mitochondrial genome-knockout cells demonstrate a dual mechanism of action for the electron transport complex I inhibitor mycothiazole.

Mycothiazole, a polyketide metabolite isolated from the marine sponge Cacospongia mycofijiensis, is a potent inhibitor of metabolic activity and mitochondrial electron transport chain complex I in sensitive cells, but other cells are relatively insensitive to the drug. Sensitive cell lines (IC(50) 0.36-13.8 nM) include HeLa, P815, RAW 264.7, MDCK, HeLa S3, 143B, 4T1, B16, and CD4/CD8 T cells. Insensitive cell lines (IC(50) 12.2-26.5 µM) include HL-60, LN18, and Jurkat. Thus, there is a 34,000-fold difference in sensitivity between HeLa and HL-60 cells. Some sensitive cell lines show a biphasic response, suggesting more than one mechanism of action. Mitochondrial genome-knockout ρ(0) cell lines are insensitive to mycothiazole, supporting a conditional mitochondrial site of action. Mycothiazole is cytostatic rather than cytotoxic in sensitive cells, has a long lag period of about 12 h, and unlike the complex I inhibitor, rotenone, does not cause G(2)/M cell cycle arrest. Mycothiazole decreases, rather than increases the levels of reactive oxygen species after 24 h. It is concluded that the cytostatic inhibitory effects of mycothiazole on mitochondrial electron transport function in sensitive cell lines may depend on a pre-activation step that is absent in insensitive cell lines with intact mitochondria, and that a second lower-affinity cytotoxic target may also be involved in the metabolic and growth inhibition of cells.

Analogs of N'-hydroxy-N-(4H,5H-naphtho[1,2-d]thiazol-2-yl)methanimidamide inhibit Mycobacterium tuberculosis methionine aminopeptidases.

Our previous target validation studies established that inhibition of methionine aminopeptidases (MtMetAP, type 1a and 1c) from Mycobacterium tuberculosis (Mtb) is an effective approach to suppress Mtb growth in culture. A novel class of MtMetAP1c inhibitors comprising of N'-hydroxy-N-(4H,5H-naphtho[1,2-d]thiazol-2-yl)methanimidamide (4c) was uncovered through a high-throughput screen (HTS). A systematic structure-activity relationship study (SAR) yielded variants of the hit, 4b, 4h, and 4k, bearing modified A- and B-rings as potent inhibitors of both MtMetAPs. Except methanimidamide 4h that showed a moderate Mtb inhibition, a desirable minimum inhibitory concentration (MIC) was not obtained with the current set of MtMetAP inhibitors. However, the SAR data generated thus far may prove valuable for further tuning of this class of inhibitors as effective anti-tuberculosis agents.

Factors associated with suicide attempts among injection drug users.

Injection drug users (IDUs) entering an urban low-threshold opiate detoxification unit in Munich, Germany, between 1991 and 1997 were asked for lifetime suicide attempts and administered a questionnaire, including sociodemographic, IDU-related, and drug user treatment related variables. Among 1,049 participants, 20% had ever tried to commit suicide. In the logistic regression, female gender, older age, drug user treatment experience, lack of drug user counseling, and emergency treatment were associated with attempted suicide. Regular screening for suicidal behavior in IDUs should be undertaken with a special focus to mention risk factors. The study's limitations are noted. This study was partially sponsored by the German Ministry of Health.