Chemistry-First Approach for Nomination of Personalized Treatment in Lung Cancer.

Diversity in the genetic lesions that cause cancer is extreme. In consequence, a pressing challenge is the development of drugs that target patient-specific disease mechanisms. To address this challenge, we employed a chemistry-first discovery paradigm for de novo identification of druggable targets linked to robust patient selection hypotheses. In particular, a 200,000 compound diversity-oriented chemical library was profiled across a heavily annotated test-bed of >100 cellular models representative of the diverse and characteristic somatic lesions for lung cancer. This approach led to the delineation of 171 chemical-genetic associations, shedding light on the targetability of mechanistic vulnerabilities corresponding to a range of oncogenotypes present in patient populations lacking effective therapy. Chemically addressable addictions to ciliogenesis in TTC21B mutants and GLUT8-dependent serine biosynthesis in KRAS/KEAP1 double mutants are prominent examples. These observations indicate a wealth of actionable opportunities within the complex molecular etiology of cancer.

High-throughput functional annotation of natural products by integrated activity profiling.

Determining mechanism of action (MOA) is one of the biggest challenges in natural products discovery. Here, we report a comprehensive platform that uses Similarity Network Fusion (SNF) to improve MOA predictions by integrating data from the cytological profiling high-content imaging platform and the gene expression platform Functional Signature Ontology, and pairs these data with untargeted metabolomics analysis for de novo bioactive compound discovery. The predictive value of the integrative approach was assessed using a library of target-annotated small molecules as benchmarks. Using Kolmogorov-Smirnov (KS) tests to compare in-class to out-of-class similarity, we found that SNF retains the ability to identify significant in-class similarity across a diverse set of target classes, and could find target classes not detectable in either platform alone. This confirmed that integration of expression-based and image-based phenotypes can accurately report on MOA. Furthermore, we integrated untargeted metabolomics of complex natural product fractions with the SNF network to map biological signatures to specific metabolites. Three examples are presented where SNF coupled with metabolomics was used to directly functionally characterize natural products and accelerate identification of bioactive metabolites, including the discovery of the azoxy-containing biaryl compounds parkamycins A and B. Our results support SNF integration of multiple phenotypic screening approaches along with untargeted metabolomics as a powerful approach for advancing natural products drug discovery.

Nonenzymatic Reactions in Natural Product Formation.

Biosynthetic mechanisms of natural products primarily depend on systems of protein catalysts. However, within the field of biosynthesis, there are cases in which the inherent chemical reactivity of metabolic intermediates and substrates evades the involvement of enzymes. These reactions are difficult to characterize based on their reactivity and occlusion within the milieu of the cellular environment. As we continue to build a strong foundation for how microbes and higher organisms produce natural products, therein lies a need for understanding how protein independent or nonenzymatic biosynthetic steps can occur. We have classified such reactions into four categories: intramolecular, multicomponent, tailoring, and light-induced reactions. Intramolecular reactions is one of the most well studied in the context of biomimetic synthesis, consisting of cyclizations and cycloadditions due to the innate reactivity of the intermediates. There are two subclasses that make up multicomponent reactions, one being homologous multicomponent reactions which results in dimeric and pseudodimeric natural products, and the other being heterologous multicomponent reactions, where two or more precursors from independent biosynthetic pathways undergo a variety of reactions to produce the mature natural product. The third type of reaction discussed are tailoring reactions, where postmodifications occur on the natural products after the biosynthetic machinery is completed. The last category consists of light-induced reactions involving ecologically relevant UV light rather than high intensity UV irradiation that is traditionally used in synthetic chemistry. This review will cover recent nonenzymatic biosynthetic mechanisms and include sources for those reviewed previously.

RNA polymerase III detects cytosolic DNA and induces type I interferons through the RIG-I pathway.

Type I interferons (IFNs) are important for antiviral and autoimmune responses. Retinoic acid-induced gene I (RIG-I) and mitochondrial antiviral signaling (MAVS) proteins mediate IFN production in response to cytosolic double-stranded RNA or single-stranded RNA containing 5'-triphosphate (5'-ppp). Cytosolic B form double-stranded DNA, such as poly(dA-dT)*poly(dA-dT) [poly(dA-dT)], can also induce IFN-beta, but the underlying mechanism is unknown. Here, we show that the cytosolic poly(dA-dT) DNA is converted into 5'-ppp RNA to induce IFN-beta through the RIG-I pathway. Biochemical purification led to the identification of DNA-dependent RNA polymerase III (Pol-III) as the enzyme responsible for synthesizing 5'-ppp RNA from the poly(dA-dT) template. Inhibition of RNA Pol-III prevents IFN-beta induction by transfection of DNA or infection with DNA viruses. Furthermore, Pol-III inhibition abrogates IFN-beta induction by the intracellular bacterium Legionella pneumophila and promotes the bacterial growth. These results suggest that RNA Pol-III is a cytosolic DNA sensor involved in innate immune responses.

Modulation of in Vitro SARS-CoV-2 Infection by Stephania tetrandra and Its Alkaloid Constituents.

Botanical natural products have been widely consumed for their purported usefulness against COVID-19. Here, six botanical species from multiple sources and 173 isolated natural product compounds were screened for blockade of wild-type (WT) SARS-CoV-2 infection in human 293T epithelial cells overexpressing ACE-2 and TMPRSS2 protease (293TAT). Antiviral activity was demonstrated by an extract from Stephania tetrandra. Extract fractionation, liquid chromatography-mass spectrometry (LC-MS), antiviral assays, and computational analyses revealed that the alkaloid fraction and purified alkaloids tetrandrine, fangchinoline, and cepharanthine inhibited WT SARS-CoV-2 infection. The alkaloids and alkaloid fraction also inhibited the delta variant of concern but not WT SARS-CoV-2 in VeroAT cells. Membrane permeability assays demonstrate that the alkaloids are biologically available, although fangchinoline showed lower permeability than tetrandrine. At high concentrations, the extract, alkaloid fractions, and pure alkaloids induced phospholipidosis in 293TAT cells and less so in VeroAT cells. Gene expression profiling during virus infection suggested that alkaloid fraction and tetrandrine displayed similar effects on cellular gene expression and pathways, while fangchinoline showed distinct effects on cells. Our study demonstrates a multifaceted approach to systematically investigate the diverse activities conferred by complex botanical mixtures, their cell-context specificity, and their pleiotropic effects on biological systems.

11B and 1H-11B HMBC NMR as a Tool for Identification of a Boron-Containing Nucleoside Dimer.

Boron-containing compounds are commonly used in synthetic chemistry and are known to play important roles in biology. Despite the widespread relevance of boronated compounds, there have been limited methods to discover, characterize, and study them. Here, we describe the use of 11B NMR, including 1H-11B HMBC, for the isolation and characterization of the boron-containing natural product diadenosine borate. Utilizing synthetic standards, we optimized coupling parameters for 1H-11B HMBC experiments to allow for the analysis of small quantities (∼1 mg) of boron-containing compounds. This work can facilitate the broader application of 11B NMR to the study of boron in a range of applications, from synthetic chemistry to the role of boron in naturally occurring systems.

Biomimetic Total Synthesis and Investigation of the Non-Enzymatic Chemistry of Oxazinin A.

We report the first total synthesis of an antimycobacterial natural product oxazinin A that takes advantage of a multi-component cascade reaction of anthranilic acid and a precursor polyketide containing an aldehyde. The route utilized for the synthesis of the pseudodimeric oxazinin A validates a previously proposed biosynthetic mechanism, invoking a non-enzymatic pathway to the complex molecule. We found a 76 : 10 : 9 : 5 ratio of oxazinin diastereomers from the synthetic cascade, which is an identical match to that found in the fermentation media from the fungus Eurotiomycetes 110162. Further investigation of the non-enzymatic formation of oxazinin A using 1 H-15 N HMBC NMR spectroscopy allowed for a plausible determination of the stepwise mechanism. The developed route is highly amenable for the synthesis of diverse sets of analogs around the oxazinin scaffold to study structure-activity relationships (SAR).

Improving natural product research translation: From source to clinical trial.

While great interest in health effects of natural product (NP) including dietary supplements and foods persists, promising preclinical NP research is not consistently translating into actionable clinical trial (CT) outcomes. Generally considered the gold standard for assessing safety and efficacy, CTs, especially phase III CTs, are costly and require rigorous planning to optimize the value of the information obtained. More effective bridging from NP research to CT was the goal of a September, 2018 transdisciplinary workshop. Participants emphasized that replicability and likelihood of successful translation depend on rigor in experimental design, interpretation, and reporting across the continuum of NP research. Discussions spanned good practices for NP characterization and quality control; use and interpretation of models (computational through in vivo) with strong clinical predictive validity; controls for experimental artefacts, especially for in vitro interrogation of bioactivity and mechanisms of action; rigorous assessment and interpretation of prior research; transparency in all reporting; and prioritization of research questions. Natural product clinical trials prioritized based on rigorous, convergent supporting data and current public health needs are most likely to be informative and ultimately affect public health. Thoughtful, coordinated implementation of these practices should enhance the knowledge gained from future NP research.

Strategies and Approaches for Discovery of Small Molecule Disruptors of Biofilm Physiology.

Biofilms, the predominant growth mode of microorganisms, pose a significant risk to human health. The protective biofilm matrix, typically composed of exopolysaccharides, proteins, nucleic acids, and lipids, combined with biofilm-grown bacteria's heterogenous physiology, leads to enhanced fitness and tolerance to traditional methods for treatment. There is a need to identify biofilm inhibitors using diverse approaches and targeting different stages of biofilm formation. This review discusses discovery strategies that successfully identified a wide range of inhibitors and the processes used to characterize their inhibition mechanism and further improvement. Additionally, we examine the structure-activity relationship (SAR) for some of these inhibitors to optimize inhibitor activity.

The value of universally available raw NMR data for transparency, reproducibility, and integrity in natural product research.

Covering: up to 2018With contributions from the global natural product (NP) research community, and continuing the Raw Data Initiative, this review collects a comprehensive demonstration of the immense scientific value of disseminating raw nuclear magnetic resonance (NMR) data, independently of, and in parallel with, classical publishing outlets. A comprehensive compilation of historic to present-day cases as well as contemporary and future applications show that addressing the urgent need for a repository of publicly accessible raw NMR data has the potential to transform natural products (NPs) and associated fields of chemical and biomedical research. The call for advancing open sharing mechanisms for raw data is intended to enhance the transparency of experimental protocols, augment the reproducibility of reported outcomes, including biological studies, become a regular component of responsible research, and thereby enrich the integrity of NP research and related fields.

Correction: The value of universally available raw NMR data for transparency, reproducibility, and integrity in natural product research.

Correction for 'The value of universally available raw NMR data for transparency, reproducibility, and integrity in natural product research' by James B. McAlpine et al., Nat. Prod. Rep., 2018, DOI: .

Ikarugamycin: A Natural Product Inhibitor of Clathrin-Mediated Endocytosis.

Ikarugamycin (IKA) is a previously discovered antibiotic, which has been shown to inhibit the uptake of oxidized low-density lipoproteins in macrophages. Furthermore, several groups have previously used IKA to inhibit clathrin-mediated endocytosis (CME) in plant cell lines. However, detailed characterization of IKA has yet to be performed. Consequently, we performed biochemistry and microscopy experiments to further characterize the effects of IKA on CME. We show that IKA has an IC50 of 2.7 µm in H1299 cells and acutely inhibits CME, but not other endocytic pathways, in a panel of cell lines. Although long-term incubation with IKA has cytotoxic effects, the short-term inhibitory effects on CME are reversible. Thus, IKA can be a useful tool for probing routes of endocytic trafficking.

Metabolite Regulation of Nuclear Localization of Carbohydrate-response Element-binding Protein (ChREBP): ROLE OF AMP AS AN ALLOSTERIC INHIBITOR.

The carbohydrate-response element-binding protein (ChREBP) is a glucose-responsive transcription factor that plays an essential role in converting excess carbohydrate to fat storage in the liver. In response to glucose levels, ChREBP is regulated by nuclear/cytosol trafficking via interaction with 14-3-3 proteins, CRM-1 (exportin-1 or XPO-1), or importins. Nuclear localization of ChREBP was rapidly inhibited when incubated in branched-chain α-ketoacids, saturated and unsaturated fatty acids, or 5-aminoimidazole-4-carboxamide ribonucleotide. Here, we discovered that protein-free extracts of high fat-fed livers contained, in addition to ketone bodies, a new metabolite, identified as AMP, which specifically activates the interaction between ChREBP and 14-3-3. The crystal structure showed that AMP binds directly to the N terminus of ChREBP-α2 helix. Our results suggest that AMP inhibits the nuclear localization of ChREBP through an allosteric activation of ChREBP/14-3-3 interactions and not by activation of AMPK. AMP and ketone bodies together can therefore inhibit lipogenesis by restricting localization of ChREBP to the cytoplasm during periods of ketosis.

Mode of action and pharmacogenomic biomarkers for exceptional responders to didemnin B.

Modern cancer treatment employs many effective chemotherapeutic agents originally discovered from natural sources. The cyclic depsipeptide didemnin B has demonstrated impressive anticancer activity in preclinical models. Clinical use has been approved but is limited by sparse patient responses combined with toxicity risk and an unclear mechanism of action. From a broad-scale effort to match antineoplastic natural products to their cellular activities, we found that didemnin B selectively induces rapid and wholesale apoptosis through dual inhibition of PPT1 and EEF1A1. Furthermore, empirical discovery of a small panel of exceptional responders to didemnin B allowed the generation of a regularized regression model to extract a sparse-feature genetic biomarker capable of predicting sensitivity to didemnin B. This may facilitate patient selection in a fashion that could enhance and expand the therapeutic application of didemnin B against neoplastic disease.

Carpatizine, a novel bridged oxazine derivative generated by non-enzymatic reactions.

Carpatizine (1), a new bridged oxazine derivative, was isolated from a marine-derived Streptomyces strain SNE-011. The structure was fully determined by spectroscopic analysis, ECD calculations and chemical methods. A plausible non-enzymatic reaction mechanism from daryamide D leading to carpatizine was presented, which was confirmed by chemical transformation.

Daryamide Analogues from a Marine-Derived Streptomyces species.

Three new cyclohexene amine derivatives, daryamides D-F (1-3), a new arylamine derivative, carpatamide D (4), and a new ornithine lactamization derivative, ornilactam A (5), were isolated from the marine-derived Streptomyces strain SNE-011. Their structures, including absolute configurations, were elucidated on the basis of spectroscopic analysis and chemical methods. The carpatamide skeleton could be considered as the biosynthetic precursor of the daryamides.

FUSION-Guided Hypothesis Development Leads to the Identification of N6,N6-Dimethyladenosine, a Marine-Derived AKT Pathway Inhibitor.

Chemicals found in nature have evolved over geological time scales to productively interact with biological molecules, and thus represent an effective resource for pharmaceutical development. Marine-derived bacteria are rich sources of chemically diverse, bioactive secondary metabolites, but harnessing this diversity for biomedical benefit is limited by challenges associated with natural product purification and determination of biochemical mechanism. Using Functional Signature Ontology (FUSION), we report the parallel isolation and characterization of a marine-derived natural product, N6,N6-dimethyladenosine, that robustly inhibits AKT signaling in a variety of non-small cell lung cancer cell lines. Upon validation of the elucidated structure by comparison with a commercially available sample, experiments were initiated to understand the small molecule's breadth of effect in a biological setting. One such experiment, a reverse phase protein array (RPPA) analysis of >50 kinases, indicated a specific cellular response to treatment. In all, leveraging the FUSION platform allowed for the rapid generation and validation of a biological mechanism of action hypothesis for an unknown natural product and permitted accelerated purification of the bioactive component from a chemically complex fraction.

Detailed Mechanistic Study of the Non-enzymatic Formation of the Discoipyrrole Family of Natural Products.

Discoipyrroles A-D (DPA-DPD) are recently discovered natural products produced by the marine bacterium Bacillus hunanensis that exhibit anticancer properties in vitro. Initial biosynthetic studies demonstrated that DPA is formed in the liquid fermentation medium of B. hunanensis from three secreted metabolites through an unknown but protein-independent mechanism. The increased identification of natural products that depend on non-enzymatic steps creates a significant need to understand how these different reactions can occur. In this work, we utilized (15)N-labeled starting materials and continuous high-sensitivity (1)H-(15)N HMBC NMR spectroscopy to resolve scarce reaction intermediates of the non-enzymatic discoipyrrole reaction as they formed in real time. This information guided supplemental experiments using (13)C- and (18)O-labeled materials to elucidate the details of DPA's non-enzymatic biosynthesis, which features a highly concerted pyrrole formation and necessary O2-mediated oxidation. We have illustrated a novel way of using isotopically enhanced two-dimensional NMR spectroscopy to interrogate reaction mechanisms as they occur. In addition, these findings add to our growing knowledge of how multicomponent non-enzymatic reactions can occur through inherently reactive bacterial metabolites.

Rifamycin Biosynthetic Congeners: Isolation and Total Synthesis of Rifsaliniketal and Total Synthesis of Salinisporamycin and Saliniketals A and B.

We describe the isolation, structure elucidation, and total synthesis of the novel marine natural product rifsaliniketal and the total synthesis of the structurally related variants salinisporamycin and saliniketals A and B. Rifsaliniketal was previously proposed, but not observed, as a diverted metabolite from a biosynthetic precursor to rifamycin S. Decarboxylation of rifamycin provides salinisporamycin, which upon truncation with loss of the naphthoquinone ring leads to saliniketals. Our synthetic strategy hinged upon a Pt(II)-catalyzed cycloisomerization of an alkynediol to set the dioxabicyclo[3.2.1]octane ring system and a fragmentation of an intermediate dihydropyranone to forge a stereochemically defined (E,Z)-dienamide unit. Multiple routes were explored to assemble fragments with high stereocontrol, an exercise that provided additional insights into acyclic stereocontrol during stereochemically complex fragment-assembly processes. The resulting 11-14 step synthesis of saliniketals then enabled us to explore strategies for the synthesis and coupling of highly substituted naphthoquinones or the corresponding naphthalene fragments. Whereas direct coupling with naphthoquinone fragments proved unsuccessful, both amidation and C-N bond formation tactics with the more electron-rich naphthalene congeners provided an efficient means to complete the first total synthesis of rifsaliniketal and salinisporamycin.

Discovery, Characterization, and Analogue Synthesis of Bohemamine Dimers Generated by Non-enzymatic Biosynthesis.

Dibohemamines A-C (5-7), three new dimeric bohemamine analogues dimerized through a methylene group, were isolated from a marine-derived Streptomyces spinoverrucosus. The structures determined by spectroscopic analysis were confirmed through the semi-synthetic derivatization of monomeric bohemamines and formaldehyde. These reactions, which could occur under mild conditions, together with the detection of formaldehyde in the culture, revealed that this dimerization is a non-enzymatic process. In addition to the unique dimerization of the dibohemamines, dibohemamines B and C were found to have nm cytotoxicity against the non-small cell-lung cancer cell line A549. In view of the potent cytotoxicity of compounds 6 and 7, a small library of bohemamine analogues was generated for biological evaluation by utilizing a series of aryl and alkyl aldehydes.