Metabolomics-Guided Discovery, Isolation, Structure Elucidation, and Bioactivity of Myropeptins C-E from Myrothecium inundatum.

The saprotrophic filamentous fungus Myrothecium inundatum represents a chemically underexplored ascomycete with a high number of putative biosynthetic gene clusters in its genome. Here, we present new linear lipopeptides from nongenetic gene activation experiments using nutrient and salt variations. Metabolomics studies revealed four myropeptins, and structural analyses by NMR, HRMS, Marfey's analysis, and ECD assessment for their helical properties established their absolute configuration. A myropeptin biosynthetic gene cluster in the genome was identified. The myropeptins exhibit general nonspecific toxicity against all cancer cell lines in the NCI-60 panel, larval zebrafish with EC50 concentrations of 5-30 µM, and pathogenic bacteria and fungi (MICs of 4-32 µg/mL against multidrug-resistant S. aureus and C. auris). In vitro hemolysis, cell viability, and ionophore assays indicate that the myropeptins target mitochondrial and cellular membranes, inducing cell depolarization and cell death. The toxic activity is modulated by the length of the lipid side chain, which provides valuable insight into their structure-activity relationships.

First trans-eunicellane terpene synthase in bacteria.

Terpenoids are the largest family of natural products, but prokaryotes are vastly underrepresented in this chemical space. However, genomics supports vast untapped biosynthetic potential for terpenoids in bacteria. We discovered the first trans-eunicellane terpene synthase (TS), AlbS from Streptomyces albireticuli NRRL B-1670, in nature. Mutagenesis, deuterium labeling studies, and quantum chemical calculations provided extensive support for its cyclization mechanism. In addition, parallel stereospecific labeling studies with Bnd4, a cis-eunicellane TS, revealed a key mechanistic distinction between these two enzymes. AlbS highlights bacteria as a valuable source of novel terpenoids, expands our understanding of the eunicellane family of natural products and the enzymes that biosynthesize them, and provides a model system to address fundamental questions about the chemistry of 6,10-bicyclic ring systems.

High-throughput infrared spectroscopy for quantification of peptides in drug discovery.

Peptides have gained an increasing importance in drug discovery as potential therapeutics. Discovery efforts toward finding new, efficacious peptide-based therapeutics have increased the throughput of peptide development, allowing the rapid generation of unique and pure peptide samples. However, high-throughput analysis of peptides may be still challenging and can encumber a high-throughput drug discovery campaign. We report herein a fit-for-purpose method to quantify peptide concentrations using high-throughput infrared spectroscopy (HT-IR). Through the development of this method, multiple critical method parameters were optimized including solvent composition, droplet deposition size, plate drying procedures, sample concentration, and internal standard. The relative absorbance of the amide region (1600-1750 cm-1) to the internal standard, K3Fe(CN)6 (2140 cm-1), was determined to be most effective at providing lowest interference for measuring peptide concentration. The best sample deposition was achieved by dissolving samples in a 50:50 v/v allyl alcohol/water mixture. The developed method was used on 96-well plates and analyzed at a rate of 22 min per plate. Calibration curves to measure sample concentration versus response relationship displayed sufficient linearity (R2 > 0.95). The repeatability and scope of detection was demonstrated with eighteen peptide samples that were measured with most values below 20% relative standard deviation. The linear dynamic range of the method was determined to be between 1 and 5 mg/mL. This developed HT-IR methodology could be a useful tool in peptide drug candidate lead identification and optimization processes.

Rapid antibody conformational screening by matrix-assisted laser desorption/ionization hydrogen-deuterium exchange mass spectrometry.

Recent advances in the field of cancer biology have accelerated the discovery and development of novel biopharmaceuticals. At the forefront of these drug development efforts are high-throughput screening, compressed timelines, and limited sample quantities, all characteristic of the discovery space. To meet program targets, large numbers of protein variants must be produced, screened, and characterized, presenting a daunting analytical challenge. Additionally, the higher-order structure is paramount for protein function and must be monitored as a critical quality attribute. Matrix-assisted laser desorption/ionization mass spectrometry has been utilized as an ultra-fast, automatable, sample-sparing analytical tool for biomolecules. Our group has published applications integrating hydrogen-deuterium exchange mass spectrometry with matrix-assisted laser desorption/ionization mass spectrometry for the rapid conformational characterization of small proteins, the current work expands this application to monoclonal and bi-specific antibodies. This study demonstrates the ability of the methodology, matrix-assisted laser desorption/ionization hydrogen-deuterium exchange mass spectrometry, to detect conformational differences between bi-specific antibodies from different expression hosts. These conformational differences were validated by orthogonal techniques including circular dichroism, nuclear magnetic resonance, and size-exclusion chromatography hydrogen-deuterium exchange mass spectrometry. This work demonstrates the utility of applying the developed methodology as a rapid conformational screening tool to triage samples for further analytical characterization.

Structure-Guided Discovery of Aminoquinazolines as Brain-Penetrant and Selective LRRK2 Inhibitors.

The leucine-rich repeat kinase 2 (LRRK2) protein has been genetically and functionally linked to Parkinson's disease (PD), a disabling and progressive neurodegenerative disorder whose current therapies are limited in scope and efficacy. In this report, we describe a rigorous hit-to-lead optimization campaign supported by structural enablement, which culminated in the discovery of brain-penetrant, candidate-quality molecules as represented by compounds 22 and 24. These compounds exhibit remarkable selectivity against the kinome and offer good oral bioavailability and low projected human doses. Furthermore, they showcase the implementation of stereochemical design elements that serve to enable a potency- and selectivity-enhancing increase in polarity and hydrogen bond donor (HBD) count while maintaining a central nervous system-friendly profile typified by low levels of transporter-mediated efflux and encouraging brain penetration in preclinical models.

Interrogation of solution conformation of complex macrocyclic peptides utilizing a combined SEC-HDX-MS, circular dichroism, and NMR workflow.

Recent technological and synthetic advances have led to a resurgence in the exploration of peptides as potential therapeutics. Understanding peptide conformation in both free and protein-bound states remains one of the most critical areas for successful development of peptide drugs. In this study it was demonstrated that the combination of Size-Exclusion Chromatography with Hydrogen-Deuterium Exchange Mass Spectrometry (SEC-HDX-MS) and Circular Dichroism Spectroscopy (CD) can be used to guide the selection of peptides for further NMR analysis. Moreover, the insights from this workflow guide the choice of the best biologically relevant conditions for NMR conformational studies of peptide ligands in a free state in solution. Combined information about solution conformation character and stability across temperatures and co-solvent compositions greatly expedites selection of optimal conditions for NMR analysis. In total, the combination of SEC-HDX-MS, CD, and NMR into a single complementary workflow greatly accelerates conformational analysis of peptides in the drug discovery lead optimization process.

Comprehensive Strategies to Bicyclic Prolines: Applications in the Synthesis of Potent Arginase Inhibitors.

Comprehensive synthetic strategies afforded a diverse set of structurally unique bicyclic proline-containing arginase inhibitors with a high degree of three-dimensionality. The analogs that favored the Cγ-exo conformation of the proline improved the arginase potency over the initial lead. The novel synthetic strategies reported here not only enable access to previously unknown stereochemically complex proline derivatives but also provide a foundation for the future synthesis of bicyclic proline analogs, which incorporate inherent three-dimensional character into building blocks, medicine, and catalysts and could have a profound impact on the conformation of proline-containing peptides and macrocycles.

Precursor-Directed Biosynthesis of Aminofulvenes: New Chalanilines from Endophytic Fungus Chalara sp.

The plant endophyte Chalara sp. is able to biotransform the epigenetic modifier vorinostat to form unique, aniline-containing polyketides named chalanilines. Here, we sought to expand the chemical diversity of chalaniline A-type molecules by changing the aniline moiety in the precursor vorinostat. In total, twenty-three different vorinostat analogs were prepared via two-step synthesis, and nineteen were incorporated by the fungus into polyketides. The highest yielding substrates were selected for large-scale precursor-directed biosynthesis and five novel compounds, including two fluorinated chalanilines, were isolated, purified, and structurally characterized. Structure elucidation relied on 1D and 2D NMR techniques and was supported by low- and high-resolution mass spectrometry. All compounds were tested for their bioactivity but were not active in antimicrobial or cell viability assays. Aminofulvene-containing natural products are rare, and this high-yielding, precursor-directed process allows for the diversification of this class of compounds.

Photomediated ring contraction of saturated heterocycles.

Saturated heterocycles are found in numerous therapeutics and bioactive natural products and are abundant in many medicinal and agrochemical compound libraries. To access new chemical space and function, many methods for functionalization on the periphery of these structures have been developed. Comparatively fewer methods are known for restructuring their core framework. Herein, we describe a visible light-mediated ring contraction of α-acylated saturated heterocycles. This unconventional transformation is orthogonal to traditional ring contractions, challenging the paradigm for diversification of heterocycles including piperidine, morpholine, thiane, tetrahydropyran, and tetrahydroisoquinoline derivatives. The success of this Norrish type II variant rests on reactivity differences between photoreactive ketone groups in specific chemical environments. This strategy was applied to late-stage remodeling of pharmaceutical derivatives, peptides, and sugars.

Brevianthrones, bianthrones from a Chinese isolate of the endophytic fungus Colletotrichum brevisporum.

Seven undescribed bianthrones, the brevianthrones, together with two known anthraquinones, were isolated from the plant-derived fungus Colletotrichum brevisporum, obtained from the plant Piper sarmentosum Roxb., collected in Guangxi, China. This is the first report of the isolation of bianthrones from the Colletotrichum genus. The structures of the compounds were elucidated by a combination of NMR and MS spectroscopic analysis, while the absolute configurations were determined by X-ray crystallography and by simulation of ECD spectra.

Discovery and Biosynthesis of a Structurally Dynamic Antibacterial Diterpenoid.

A new bicyclic diterpenoid, benditerpenoic acid, was isolated from soil-dwelling Streptomyces sp. (CL12-4). We sequenced the bacterial genome, identified the responsible biosynthetic gene cluster, verified the function of the terpene synthase, and heterologously produced the core diterpene. Comparative bioinformatics indicated this Streptomyces strain is phylogenetically unique and possesses nine terpene synthases. The absolute configurations of the new trans-fused bicyclo[8.4.0]tetradecanes were achieved by extensive spectroscopic analyses, including Mosher's analysis, J-based coupling analysis, and computations based on sparse NMR-derived experimental restraints. Interestingly, benditerpenoic acid exists in two distinct ring-flipped bicyclic conformations with a rotational barrier of ≈16 kcal mol-1 in solution. The diterpenes exhibit moderate antibacterial activity against Gram-positive bacteria including methicillin and multi-drug resistant Staphylococcus aureus. This is a rare example of an eunicellane-type diterpenoid from bacteria and the first identification of a diterpene synthase and biosynthetic gene cluster responsible for the construction of the eunicellane scaffold.

Transcriptomic, Protein-DNA Interaction, and Metabolomic Studies of VosA, VelB, and WetA in Aspergillus nidulans Asexual Spores.

In filamentous fungi, asexual development involves cellular differentiation and metabolic remodeling leading to the formation of intact asexual spores. The development of asexual spores (conidia) in Aspergillus is precisely coordinated by multiple transcription factors (TFs), including VosA, VelB, and WetA. Notably, these three TFs are essential for the structural and metabolic integrity, i.e., proper maturation, of conidia in the model fungus Aspergillus nidulans To gain mechanistic insight into the complex regulatory and interdependent roles of these TFs in asexual sporogenesis, we carried out multi-omics studies on the transcriptome, protein-DNA interactions, and primary and secondary metabolism employing A. nidulans conidia. RNA sequencing and chromatin immunoprecipitation sequencing analyses have revealed that the three TFs directly or indirectly regulate the expression of genes associated with heterotrimeric G-protein signal transduction, mitogen-activated protein (MAP) kinases, spore wall formation and structural integrity, asexual development, and primary/secondary metabolism. In addition, metabolomics analyses of wild-type and individual mutant conidia indicate that these three TFs regulate a diverse array of primary metabolites, including those in the tricarboxylic acid (TCA) cycle, certain amino acids, and trehalose, and secondary metabolites such as sterigmatocystin, emericellamide, austinol, and dehydroaustinol. In summary, WetA, VosA, and VelB play interdependent, overlapping, and distinct roles in governing morphological development and primary/secondary metabolic remodeling in Aspergillus conidia, leading to the production of vital conidia suitable for fungal proliferation and dissemination.IMPORTANCE Filamentous fungi produce a vast number of asexual spores that act as efficient propagules. Due to their infectious and/or allergenic nature, fungal spores affect our daily life. Aspergillus species produce asexual spores called conidia; their formation involves morphological development and metabolic changes, and the associated regulatory systems are coordinated by multiple transcription factors (TFs). To understand the underlying global regulatory programs and cellular outcomes associated with conidium formation, genomic and metabolomic analyses were performed in the model fungus Aspergillus nidulans Our results show that the fungus-specific WetA/VosA/VelB TFs govern the coordination of morphological and chemical developments during sporogenesis. The results of this study provide insights into the interdependent, overlapping, or distinct genetic regulatory networks necessary to produce intact asexual spores. The findings are relevant for other Aspergillus species such as the major human pathogen Aspergillus fumigatus and the aflatoxin producer Aspergillus flavus.

Biodiversity, Bioactivity, and Metabolites of High Desert Derived Oregonian Soil Bacteria.

From arid, high desert soil samples collected near Bend, Oregon, 19 unique bacteria were isolated. Each strain was identified by 16S rRNA gene sequencing, and their organic extracts were tested for antibacterial and antiproliferative activities. Noteworthy, six extracts (30 %) exhibited strong inhibition resulting in less than 50 % cell proliferation in more than one cancer cell model, tested at 10 µg/mL. Principal component analysis (PCA) of LC/MS data revealed drastic differences in the metabolic profiles found in the organic extracts of these soil bacteria. In total, fourteen potent antibacterial and/or cytotoxic metabolites were isolated via bioactivity-guided fractionation, including two new natural products: a pyrazinone containing tetrapeptide and 7-methoxy-2,3-dimethyl-4H-chromen-4-one, as well as twelve known compounds: furanonaphthoquinone I, bafilomycin C1 and D, FD-594, oligomycin A, chloramphenicol, MY12-62A, rac-sclerone, isosclerone, tunicamycin VII, tunicamycin VIII, and (6S,16S)-anthrabenzoxocinone 1.264-C.

Synthesis of Bridged Bicyclic Amines by Intramolecular Amination of Remote C-H Bonds: Synergistic Activation by Light and Heat.

The construction of complex aza-cycles is of interest to drug discovery due to the prevalence of nitrogen-containing heterocycles in pharmaceutical agents. Herein we report an intramolecular C-H amination approach to afford value-added and complexity-enriched bridged bicyclic amines. Guided by density functional theory and nuclear magnetic resonance investigations, we determined the unique roles of light and heat activation in the bicyclization mechanism. We applied both light and heat activation in a synergistic fashion, achieving gram-scale bridged aza-cycle synthesis.

N-Linked Surface Glycan Biosynthesis, Composition, Inhibition, and Function in Cnidarian-Dinoflagellate Symbiosis.

The success of symbioses between cnidarian hosts (e.g., corals and sea anemones) and micro-algal symbionts hinges on the molecular interactions that govern the establishment and maintenance of intracellular mutualisms. As a fundamental component of innate immunity, glycan-lectin interactions impact the onset of marine endosymbioses, but our understanding of the effects of cell surface glycome composition on symbiosis establishment remains limited. In this study, we examined the canonical N-glycan biosynthesis pathway in the genome of the dinoflagellate symbiont Breviolum minutum (family Symbiodiniaceae) and found it to be conserved with the exception of the transferase GlcNAc-TII (MGAT2). Using coupled liquid chromatography-mass spectrometry (LC-MS/MS), we characterized the cell surface N-glycan content of B. minutum, providing the first insight into the molecular composition of surface glycans in dinoflagellates. We then used the biosynthesis inhibitors kifunensine and swainsonine to alter the glycan composition of B. minutum. Successful high-mannose enrichment via kifunensine treatment resulted in a significant decrease in colonization of the model sea anemone Aiptasia (Exaiptasia pallida) by B. minutum. Hybrid glycan enrichment via swainsonine treatment, however, could not be confirmed and did not impact colonization. We conclude that functional Golgi processing of N-glycans is critical for maintaining appropriate cell surface glycan composition and for ensuring colonization success by B. minutum.

C─C Cleavage Approach to C─H Functionalization of Saturated Aza-Cycles.

Saturated cyclic amines (aza-cycles) are ubiquitous structural motifs found in pharmaceuticals, agrochemicals, and bioactive natural products. Given their importance, methods that directly functionalize aza-cycles are in high demand. Herein, we disclose a fundamentally different approach to functionalizing cyclic amines which relies on C─C cleavage and attendant cross-coupling. The initial functionalization step is the generation of underexplored N-fused bicyclo α-hydroxy-ß-lactams under mild, visible light conditions using a Norrish-Yang process to affect α-functionalization of saturated cyclic amines. This approach is complementary to previous methods for the C─H functionalization of aza-cycles and provides unique access to various cross-coupling adducts. In the course of these studies, we have also uncovered an orthogonal, base-promoted opening of the N-fused bicyclo α-hydroxy-ß-lactams. Computational studies have provided insight into the origin of the complementary C─C cleavage processes.

Polyketides from Marine-Derived Aspergillus porosus: Challenges and Opportunities for Determining Absolute Configuration.

Fungal natural products have inspired and enabled countless modern therapeutics. During a survey of the secondary metabolites of endophytic fungi, we found that Aspergillus porosus produces new polyketides with interesting structural features named porosuphenols A-D (1, 2, 3a, and 3b). The structural elucidation of these metabolites was performed with 1D and 2D NMR techniques, Mosher ester analysis, J-based conformational analysis, and isotope exchange studies. The absolute configuration of these compounds was determined using typical approaches including comparative analysis of experimental NMR and electronic circular dichroism spectra with DFT calculations. However, these efforts did not provide conclusive results for porosuphenol A (1). To resolve this issue, we applied a strategy in which NMR data guide the conformer search. Herein are presented the structure elucidation of porosuphenols A-D as a case study in the challenges and opportunities for determination of absolute configuration. Lastly, bioassay-guided fractionation of cytotoxic fractions resulted in the additional isolation of pimarane diterpenes, sphaeropsidin A (4), and aspergiloid E (5).

A metabolomics-guided approach to discover Fusarium graminearum metabolites after removal of a repressive histone modification.

Many secondary metabolites are produced by biosynthetic gene clusters (BGCs) that are repressed during standard growth conditions, which complicates the discovery of novel bioactive compounds. In the genus Fusarium, many BGCs reside in chromatin enriched for trimethylated histone 3 lysine 27 (H3K27me3), a modification correlated with transcriptional gene silencing. Here we report on our progress in assigning metabolites to genes by using a strain lacking the H3K27 methyltransferase, Kmt6. To guide isolation efforts, we coupled genetics to multivariate analysis of liquid chromatography-mass spectrometry (LCMS) data from both wild type and kmt6, which allowed identification of compounds previously unknown from F. graminearum. We found low molecular weight, amino acid-derived metabolites (N-ethyl anthranilic acid, N-phenethylacetamide, N-acetyltryptamine). We identified one new compound, protofusarin, as derived from fusarin biosynthesis. Similarly, we isolated large amounts of fusaristatin A, gibepyrone A, and fusarpyrones A and B, simply by using the kmt6 mutant, instead of having to optimize growth media. To increase the abundance of metabolites underrepresented in wild type, we generated kmt6 fus1 double mutants and discovered tricinolone and tricinolonoic acid, two new sesquiterpenes belonging to the tricindiol class. Our approach allows rapid visualization and analyses of the genetically induced changes in metabolite production, and discovery of new molecules by a combination of chemical and genetic dereplication. Of 22 fungal metabolites identified here, 10 compounds had not been reported from F. graminearum before. We show that activating silent metabolic pathways by mutation of a repressive chromatin modification enzyme can result in the discovery of new chemistry even in a well-studied organism, and helps to connect new or known small molecules to the BGCs responsible for their production.

Subtle Differences in Symbiont Cell Surface Glycan Profiles Do Not Explain Species-Specific Colonization Rates in a Model Cnidarian-Algal Symbiosis.

Mutualisms between cnidarian hosts and dinoflagellate endosymbionts are foundational to coral reef ecosystems. These symbioses are often re-established every generation with high specificity, but gaps remain in our understanding of the cellular mechanisms that control symbiont recognition and uptake dynamics. Here, we tested whether differences in glycan profiles among different symbiont species account for the different rates at which they initially colonize aposymbiotic polyps of the model sea anemone Aiptasia (Exaiptasia pallida). First, we used a lectin array to characterize the glycan profiles of colonizing Symbiodinium minutum (ITS2 type B1) and noncolonizing Symbiodinium pilosum (ITS2 type A2), finding subtle differences in the binding of lectins Euonymus europaeus lectin (EEL) and Urtica dioica agglutinin lectin (UDA) that distinguish between high-mannoside and hybrid-type protein linked glycans. Next, we enzymatically cleaved glycans from the surfaces of S. minutum cultures and followed their recovery using flow cytometry, establishing a 48-72 h glycan turnover rate for this species. Finally, we exposed aposymbiotic host polyps to cultured S. minutum cells masked by EEL or UDA lectins for 48 h, then measured cell densities the following day. We found no effect of glycan masking on symbiont density, providing further support to the hypothesis that glycan-lectin interactions are more important for post-phagocytic persistence of specific symbionts than they are for initial uptake. We also identified several methodological and biological factors that may limit the utility of studying glycan masking in the Aiptasia system.

Unexpected Biotransformation of the HDAC Inhibitor Vorinostat Yields Aniline-Containing Fungal Metabolites.

The diversity of genetically encoded small molecules produced by filamentous fungi remains largely unexplored, which makes these fungi an attractive source for the discovery of new compounds. However, accessing their full chemical repertoire under common laboratory culture conditions is a challenge. Epigenetic manipulation of gene expression has become a well-established tool for overcoming this obstacle. Here, we report that perturbation of the endophytic ascomycete Chalara sp. 6661, producer of the isofusidienol class of antibiotics, with the HDAC inhibitor vorinostat resulted in the production of four new modified xanthones. The structures of chalanilines A (1) and B (2) and adenosine-coupled xanthones A (3) and B (4) were determined by extensive NMR spectroscopic analyses, and the bioactivities of 1-4 were tested in antibiotic and cytotoxicity assays. Incorporation studies with deuterium-labeled vorinostat indicate that the aniline moiety in chalalanine A is derived from vorinostat itself. Our study shows that Chalara sp. is able to metabolize the HDAC inhibitor vorinostat to release aniline. This is a rare report of fungal biotransformation of the popular epigenetic modifier vorinostat into aniline-containing polyketides.