Neutral or detrimental effects of TREM2 agonist antibodies in preclinical models of Alzheimer's Disease and Multiple Sclerosis.

Human genetics and preclinical studies have identified key contributions of TREM2 to several neurodegenerative conditions, inspiring efforts to modulate TREM2 therapeutically. Here, we characterize the activities of three TREM2 agonist antibodies in multiple mixed-sex mouse models of Alzheimer's Disease (AD) pathology and remyelination. Receptor activation and downstream signaling are explored in vitro, and active dose ranges are determined in vivo based on pharmacodynamic responses from microglia. For mice bearing amyloid-ß (Aß) pathology (PS2APP) or combined Aß and tau pathology (TauPS2APP), chronic TREM2 agonist antibody treatment had limited impact on microglia engagement with pathology, overall pathology burden, or downstream neuronal damage. For mice with demyelinating injuries triggered acutely with lysolecithin, TREM2 agonist antibodies unexpectedly disrupted injury resolution. Likewise, TREM2 agonist antibodies limited myelin recovery for mice experiencing chronic demyelination from cuprizone. We highlight the contributions of dose timing and frequency across models. These results introduce important considerations for future TREM2-targeting approaches.Significance Statement Multiple TREM2 agonist antibodies are investigated in mouse models of Alzheimer's Disease and Multiple Sclerosis. Despite agonism in culture models and after acute dosing in mice, antibodies do not show benefit in overall AD pathology and worsen recovery after demyelination.

LOXL4, but not LOXL2, is the critical determinant of pathological collagen cross-linking and fibrosis in the lung.

Idiopathic pulmonary fibrosis is a progressive fibrotic disease characterized by excessive deposition of (myo)fibroblast produced collagen fibrils in alveolar areas of the lung. Lysyl oxidases (LOXs) have been proposed to be the central enzymes that catalyze the cross-linking of collagen fibers. Here, we report that, while its expression is increased in fibrotic lungs, genetic ablation of LOXL2 only leads to a modest reduction of pathological collagen cross-linking but not fibrosis in the lung. On the other hand, loss of another LOX family member, LOXL4, markedly disrupts pathological collagen cross-linking and fibrosis in the lung. Furthermore, knockout of both Loxl2 and Loxl4 does not offer any additive antifibrotic effects when compared to Loxl4 deletion only, as LOXL4 deficiency decreases the expression of other LOX family members including Loxl2. On the basis of these results, we propose that LOXL4 is the main LOX activity underlying pathological collagen cross-linking and lung fibrosis.

Bioactive lipid lysophosphatidic acid species are associated with disease progression in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive disease with significant mortality. Prognostic biomarkers to identify rapid progressors are urgently needed to improve patient management. Since the lysophosphatidic acid (LPA) pathway has been implicated in lung fibrosis in preclinical models and identified as a potential therapeutic target, we aimed to investigate if bioactive lipid LPA species could be prognostic biomarkers that predict IPF disease progression. LPAs and lipidomics were measured in baseline placebo plasma of a randomized IPF-controlled trial. The association of lipids with disease progression indices were assessed using statistical models. Compared to healthy, IPF patients had significantly higher levels of five LPAs (LPA16:0, 16:1, 18:1, 18:2, 20:4) and reduced levels of two triglycerides species (TAG48:4-FA12:0, -FA18:2) (false discovery rate < 0.05, fold change > 2). Patients with higher levels of LPAs had greater declines in diffusion capacity of carbon monoxide over 52 weeks (P < 0.01); additionally, LPA20:4-high (≥median) patients had earlier time to exacerbation compared to LPA20:4-low (

Stress-induced vesicular assemblies of dual leucine zipper kinase are signaling hubs involved in kinase activation and neurodegeneration.

Mitogen-activated protein kinases (MAPKs) drive key signaling cascades during neuronal survival and degeneration. The localization of kinases to specific subcellular compartments is a critical mechanism to locally control signaling activity and specificity upon stimulation. However, how MAPK signaling components tightly control their localization remains largely unknown. Here, we systematically analyzed the phosphorylation and membrane localization of all MAPKs expressed in dorsal root ganglia (DRG) neurons, under control and stress conditions. We found that MAP3K12/dual leucine zipper kinase (DLK) becomes phosphorylated and palmitoylated, and it is recruited to sphingomyelin-rich vesicles upon stress. Stress-induced DLK vesicle recruitment is essential for kinase activation; blocking DLK-membrane interaction inhibits downstream signaling, while DLK recruitment to ectopic subcellular structures is sufficient to induce kinase activation. We show that the localization of DLK to newly formed vesicles is essential for local signaling. Inhibition of membrane internalization blocks DLK activation and protects against neurodegeneration in DRG neurons. These data establish vesicular assemblies as dynamically regulated platforms for DLK signaling during neuronal stress responses.

NP Analyst: An Open Online Platform for Compound Activity Mapping.

Few tools exist in natural products discovery to integrate biological screening and untargeted mass spectrometry data at the library scale. Previously, we reported Compound Activity Mapping as a strategy for predicting compound bioactivity profiles directly from primary screening results on extract libraries. We now present NP Analyst, an open online platform for Compound Activity Mapping that accepts bioassay data of almost any type, and is compatible with mass spectrometry data from major instrument manufacturers via the mzML format. In addition, NP Analyst will accept processed mass spectrometry data from the MZmine 2 and GNPS open-source platforms, making it a versatile tool for integration with existing discovery workflows. We demonstrate the utility of this new tool for both the dereplication of known compounds and the discovery of novel bioactive natural products using a challenging low-resolution antimicrobial bioassay data set. This new platform is available at www.npanalyst.org.

Sansanmycin natural product analogues as potent and selective anti-mycobacterials that inhibit lipid I biosynthesis.

Tuberculosis (TB) is responsible for enormous global morbidity and mortality, and current treatment regimens rely on the use of drugs that have been in use for more than 40 years. Owing to widespread resistance to these therapies, new drugs are desperately needed to control the TB disease burden. Herein, we describe the rapid synthesis of analogues of the sansanmycin uridylpeptide natural products that represent promising new TB drug leads. The compounds exhibit potent and selective inhibition of Mycobacterium tuberculosis, the etiological agent of TB, both in vitro and intracellularly. The natural product analogues are nanomolar inhibitors of Mtb phospho-MurNAc-pentapeptide translocase, the enzyme responsible for the synthesis of lipid I in mycobacteria. This work lays the foundation for the development of uridylpeptide natural product analogues as new TB drug candidates that operate through the inhibition of peptidoglycan biosynthesis.

Piericidin A1 Blocks Yersinia Ysc Type III Secretion System Needle Assembly.

The type III secretion system (T3SS) is a bacterial virulence factor expressed by dozens of Gram-negative pathogens but largely absent from commensals. The T3SS is an attractive target for antimicrobial agents that may disarm pathogenic bacteria while leaving commensal populations intact. We previously identified piericidin A1 as an inhibitor of the Ysc T3SS in Yersinia pseudotuberculosis. Piericidins were first discovered as inhibitors of complex I of the electron transport chain in mitochondria and some bacteria. However, we found that piericidin A1 did not alter Yersinia membrane potential or inhibit flagellar motility powered by the proton motive force, indicating that the piericidin mode of action against Yersinia type III secretion is independent of complex I. Instead, piericidin A1 reduced the number of T3SS needle complexes visible by fluorescence microscopy at the bacterial surface, preventing T3SS translocator and effector protein secretion. Furthermore, piericidin A1 decreased the abundance of higher-order YscF needle subunit complexes, suggesting that piericidin A1 blocks YscF needle assembly. While expression of T3SS components in Yersinia are positively regulated by active type III secretion, the block in secretion by piericidin A1 was not accompanied by a decrease in T3SS gene expression, indicating that piericidin A1 may target a T3SS regulatory circuit. However, piericidin A1 still inhibited effector protein secretion in the absence of the T3SS regulator YopK, YopD, or YopN. Surprisingly, while piericidin A1 also inhibited the Y. enterocolitica Ysc T3SS, it did not inhibit the SPI-1 family Ysa T3SS in Y. enterocolitica or the Ysc family T3SS in Pseudomonas aeruginosa. Together, these data indicate that piericidin A1 specifically inhibits Yersinia Ysc T3SS needle assembly. IMPORTANCE The bacterial type III secretion system (T3SS) is widely used by both human and animal pathogens to cause disease yet remains incompletely understood. Deciphering how some natural products, such as the microbial metabolite piericidin, inhibit type III secretion can provide important insight into how the T3SS functions or is regulated. Taking this approach, we investigated the ability of piericidin to block T3SS function in several human pathogens. Surprisingly, piericidin selectively inhibited the Ysc family T3SS in enteropathogenic Yersinia but did not affect the function of a different T3SS within the same species. Furthermore, piericidin specifically blocked the formation of T3SS needles on the bacterial surface without altering the localization of several other T3SS components or regulation of T3SS gene expression. These data show that piericidin targets a mechanism important for needle assembly that is unique to the Yersinia Ysc T3SS.

Sloth hair as a novel source of fungi with potent anti-parasitic, anti-cancer and anti-bacterial bioactivity.

The extraordinary biological diversity of tropical forests harbors a rich chemical diversity with enormous potential as a source of novel bioactive compounds. Of particular interest are new environments for microbial discovery. Sloths--arboreal mammals commonly found in the lowland forests of Panama--carry a wide variety of micro- and macro-organisms on their coarse outer hair. Here we report for the first time the isolation of diverse and bioactive strains of fungi from sloth hair, and their taxonomic placement. Eighty-four isolates of fungi were obtained in culture from the surface of hair that was collected from living three-toed sloths (Bradypus variegatus, Bradypodidae) in Soberanía National Park, Republic of Panama. Phylogenetic analyses revealed a diverse group of Ascomycota belonging to 28 distinct operational taxonomic units (OTUs), several of which are divergent from previously known taxa. Seventy-four isolates were cultivated in liquid broth and crude extracts were tested for bioactivity in vitro. We found a broad range of activities against strains of the parasites that cause malaria (Plasmodium falciparum) and Chagas disease (Trypanosoma cruzi), and against the human breast cancer cell line MCF-7. Fifty fungal extracts were tested for antibacterial activity in a new antibiotic profile screen called BioMAP; of these, 20 were active against at least one bacterial strain, and one had an unusual pattern of bioactivity against Gram-negative bacteria that suggests a potentially new mode of action. Together our results reveal the importance of exploring novel environments for bioactive fungi, and demonstrate for the first time the taxonomic composition and bioactivity of fungi from sloth hair.

MS/MS-based networking and peptidogenomics guided genome mining revealed the stenothricin gene cluster in Streptomyces roseosporus.

Most (75%) of the anti-infectives that save countless lives and enormously improve quality of life originate from microbes found in nature. Herein, we described a global visualization of the detectable molecules produced from a single microorganism, which we define as the 'molecular network' of that organism, followed by studies to characterize the cellular effects of antibacterial molecules. We demonstrate that Streptomyces roseosporus produces at least four non-ribosomal peptide synthetase-derived molecular families and their gene subnetworks (daptomycin, arylomycin, napsamycin and stenothricin) were identified with different modes of action. A number of previously unreported analogs involving truncation, glycosylation, hydrolysis and biosynthetic intermediates and/or shunt products were also captured and visualized by creation of a map through MS/MS networking. The diversity of antibacterial compounds produced by S. roseosporus highlights the importance of developing new approaches to characterize the molecular capacity of an organism in a more global manner. This allows one to more deeply interrogate the biosynthetic capacities of microorganisms with the goal to streamline the discovery pipeline for biotechnological applications in agriculture and medicine. This is a contribution to a special issue to honor Chris Walsh's amazing career.

An NF-κB-based high-throughput screen identifies piericidins as inhibitors of the Yersinia pseudotuberculosis type III secretion system.

The type III secretion system (T3SS) is a bacterial appendage used by dozens of Gram-negative pathogens to subvert host defenses and cause disease, making it an ideal target for pathogen-specific antimicrobials. Here, we report the discovery and initial characterization of two related natural products with T3SS-inhibitory activity that were derived from a marine actinobacterium. Bacterial extracts containing piericidin A1 and the piericidin derivative Mer-A 2026B inhibited Yersinia pseudotuberculosis from triggering T3SS-dependent activation of the host transcription factor NF-κB in HEK293T cells but were not toxic to mammalian cells. As the Yersinia T3SS must be functional in order to trigger NF-κB activation, these data indicate that piericidin A1 and Mer-A 2026B block T3SS function. Consistent with this, purified piericidin A1 and Mer-A 2026B dose-dependently inhibited translocation of the Y. pseudotuberculosis T3SS effector protein YopM inside CHO cells. In contrast, neither compound perturbed bacterial growth in vitro, indicating that piericidin A1 and Mer-A 2026B do not function as general antibiotics in Yersinia. In addition, when Yersinia was incubated under T3SS-inducing culture conditions in the absence of host cells, Mer-A 2026B and piericidin A1 inhibited secretion of T3SS cargo as effectively as or better than several previously described T3SS inhibitors, such as MBX-1641 and aurodox. This suggests that Mer-A 2026B and piericidin A1 do not block type III secretion by blocking the bacterium-host cell interaction, but rather inhibit an earlier stage, such as T3SS needle assembly. In summary, the marine-derived natural products Mer-A 2026B and piericidin A1 possess previously uncharacterized activity against the bacterial T3SS.

Molecular networking as a dereplication strategy.

A major goal in natural product discovery programs is to rapidly dereplicate known entities from complex biological extracts. We demonstrate here that molecular networking, an approach that organizes MS/MS data based on chemical similarity, is a powerful complement to traditional dereplication strategies. Successful dereplication with molecular networks requires MS/MS spectra of the natural product mixture along with MS/MS spectra of known standards, synthetic compounds, or well-characterized organisms, preferably organized into robust databases. This approach can accommodate different ionization platforms, enabling cross correlations of MS/MS data from ambient ionization, direct infusion, and LC-based methods. Molecular networking not only dereplicates known molecules from complex mixtures, it also captures related analogues, a challenge for many other dereplication strategies. To illustrate its utility as a dereplication tool, we apply mass spectrometry-based molecular networking to a diverse array of marine and terrestrial microbial samples, illustrating the dereplication of 58 molecules including analogues.

Development of quinoline-based disruptors of biofilm formation against Vibrio cholerae.

Biofilm formation is a major cause of bacterial persistence in nosocomial infections, leading to extended treatment times and increased rates of morbidity and mortality. Despite this, there are currently no biofilm inhibitors approved for clinical use. The synthesis and biological evaluation of a library of amino alcohol quinolines as lead compounds for the disruption of biofilm formation in Vibrio cholerae is now reported. Application of selective metal-halogen exchange chemistry installed both stereocenters in one step, to afford a simpler scaffold than the initial lead molecule, with an EC50 < 10 µM.

Development of antibiotic activity profile screening for the classification and discovery of natural product antibiotics.

Despite recognition of the looming antibiotic crisis by healthcare professionals, the number of new antibiotics reaching the clinic continues to decline sharply. This study aimed to establish an antibiotic profiling strategy using a panel of clinically relevant bacterial strains to create unique biological fingerprints for all major classes of antibiotics. Antibiotic mode of action profile (BioMAP) screening has been shown to effectively cluster antibiotics by structural class based on these fingerprints. Using this approach, we have accurately predicted the presence of known antibiotics in natural product extracts and have discovered a naphthoquinone-based antibiotic from our marine natural product library that possesses a unique carbon skeleton. We have demonstrated that bioactivity fingerprinting is a successful strategy for profiling antibiotic lead compounds and that BioMAP can be applied to the discovery of new natural product antibiotics leads.

Examining the fish microbiome: vertebrate-derived bacteria as an environmental niche for the discovery of unique marine natural products.

Historically, marine invertebrates have been a prolific source of unique natural products, with a diverse array of biological activities. Recent studies of invertebrate-associated microbial communities are revealing microorganisms as the true producers of many of these compounds. Inspired by the human microbiome project, which has highlighted the human intestine as a unique microenvironment in terms of microbial diversity, we elected to examine the bacterial communities of fish intestines (which we have termed the fish microbiome) as a new source of microbial and biosynthetic diversity for natural products discovery. To test the hypothesis that the fish microbiome contains microorganisms with unique capacity for biosynthesizing natural products, we examined six species of fish through a combination of dissection and culture-dependent evaluation of intestinal microbial communities. Using isolation media designed to enrich for marine Actinobacteria, we have found three main clades that show taxonomic divergence from known strains, several of which are previously uncultured. Extracts from these strains exhibit a wide range of activities against both gram-positive and gram-negative human pathogens, as well as several fish pathogens. Exploration of one of these extracts has identified the novel bioactive lipid sebastenoic acid as an anti-microbial agent, with activity against Staphylococcus aureus, Bacillus subtilis, Enterococcus faecium, and Vibrio mimicus.