Benzo[a]pyrene toxicokinetics in humans following dietary supplementation with 3,3'-diindolylmethane (DIM) or Brussels sprouts.

Utilizing the atto-zeptomole sensitivity of UPLC-accelerator mass spectrometry (UPLC-AMS), we previously demonstrated significant first-pass metabolism following escalating (25-250 ng) oral micro-dosing in humans of [14C]-benzo[a]pyrene ([14C]-BaP). The present study examines the potential for supplementation with Brussels sprouts (BS) or 3,3'-diindolylmethane (DIM) to alter plasma levels of [14C]-BaP and metabolites over a 48-h period following micro-dosing with 50 ng (5.4 nCi) [14C]-BaP. Volunteers were dosed with [14C]-BaP following fourteen days on a cruciferous vegetable restricted diet, or the same diet supplemented for seven days with 50 g of BS or 300 mg of BR-DIM® prior to dosing. BS or DIM reduced total [14C] recovered from plasma by 56-67% relative to non-intervention. Dietary supplementation with DIM markedly increased Tmax and reduced Cmax for [14C]-BaP indicative of slower absorption. Both dietary treatments significantly reduced Cmax values of four downstream BaP metabolites, consistent with delaying BaP absorption. Dietary treatments also appeared to reduce the T1/2 and the plasma AUC(0,∞) for Unknown Metabolite C, indicating some effect in accelerating clearance of this metabolite. Toxicokinetic constants for other metabolites followed the pattern for [14C]-BaP (metabolite profiles remained relatively consistent) and non-compartmental analysis did not indicate other significant alterations. Significant amounts of metabolites in plasma were at the bay region of [14C]-BaP irrespective of treatment. Although the number of subjects and large interindividual variation are limitations of this study, it represents the first human trial showing dietary intervention altering toxicokinetics of a defined dose of a known human carcinogen.

Thorectidiol A Isolated from the Marine Sponge Dactylospongia elegans Disrupts Interactions of the SARS-CoV-2 Spike Receptor Binding Domain with the Host ACE2 Receptor.

Thorectidiols isolated from the marine sponge Dactylospongia elegans (family Thorectidae, order Dictyoceratida) collected in Papua New Guinea are a family of symmetrical and unsymmetrical dimeric biphenyl meroterpenoid stereoisomers presumed to be products of oxidative phenol coupling of a co-occurring racemic monomer, thorectidol (3). One member of the family, thorectidiol A (1), has been isolated in its natural form, and its structure has been elucidated by analysis of NMR, MS, and ECD data. Acetylation of the sponge extract facilitated isolation of additional thorectidiol diacetate stereoisomers and the isolation of the racemic monomer thorectidol acetate (6). Racemic thorectidiol A (1) showed selective inhibition of the SARS-CoV-2 spike receptor binding domain (RBD) interaction with the host ACE2 receptor with an IC50 = 1.0 ± 0.7 µM.

Translating dosimetry of Dibenzo[def,p]chrysene (DBC) and metabolites across dose and species using physiologically based pharmacokinetic (PBPK) modeling.

Dibenzo[def,p]chrysene (DBC) is an environmental polycyclic aromatic hydrocarbon (PAH) that causes tumors in mice and has been classified as a probable human carcinogen by the International Agency for Research on Cancer. Animal toxicity studies often utilize higher doses than are found in relevant human exposures. Additionally, like many PAHs, DBC requires metabolic bioactivation to form the ultimate toxicant, and species differences in DBC and DBC metabolite metabolism have been observed. To understand the implications of dose and species differences, a physiologically based pharmacokinetic model (PBPK) for DBC and major metabolites was developed in mice and humans. Metabolism parameters used in the model were obtained from experimental in vitro metabolism assays using mice and human hepatic microsomes. PBPK model simulations were evaluated against mice dosed with 15 mg/kg DBC by oral gavage and human volunteers orally microdosed with 29 ng of DBC. DBC and its primary metabolite DBC-11,12-diol were measured in blood of mice and humans, while in urine, the majority of DBC metabolites were obeserved as conjugated DBC-11,12-diol, conjugated DBC tetrols, and unconjugated DBC tetrols. The PBPK model was able to predict the time course concentrations of DBC, DBC-11,12-diol, and other DBC metabolites in blood and urine of human volunteers and mice with reasonable accuracy. Agreement between model simulations and measured pharmacokinetic data in mice and human studies demonstrate the success and versatility of our model for interspecies extrapolation and applicability for different doses. Furthermore, our simulations show that internal dose metrics used for risk assessment do not necessarily scale allometrically, and that PBPK modeling provides a reliable approach to appropriately account for interspecies differences in metabolism and physiology.

Current Approaches to COVID-19 Drug Discovery and Safety Assessment.

Various methods currently being explored to treat COVID-19 were discussed during a symposium at the Fall 2022 ACS conference. These methods included the inhibiting of immune responses and viral replication pathways as well as repurposing known drugs.

Ansellone J, a Potent in Vitro and ex Vivo HIV-1 Latency Reversal Agent Isolated from a Phorbas sp. Marine Sponge.

Five new minor sesterterpenoids, ansellones H (4), I (5), J (6), and K (7) and phorone C (8), have been isolated from a Phorbas sp. marine sponge collected in British Columbia. Their structures have been elucidated by detailed analysis of NMR and MS data. Ansellone J (6) and phorone C (8) are potent in vitro HIV-1 latency reversal agents that are more potent than the reference compound and control protein kinase C activator prostratin (3). The most potent Phorbas sesterterpenoid, ansellone J (6), was evaluated for HIV latency reversal in a primary cell context using CD4+ T cells obtained directly from four combination antiretroviral therapy-suppressed donors with HIV. To a first approximation, ansellone J (6) induced HIV latency reversal at levels similar to prostratin (3) ex vivo, but at a 10-fold lower concentration.

Natural Products Produced in Culture by Biosynthetically Talented Salinispora arenicola Strains Isolated from Northeastern and South Pacific Marine Sediments.

Laboratory cultures of two 'biosynthetically talented' bacterial strains harvested from tropical and temperate Pacific Ocean sediment habitats were examined for the production of new natural products. Cultures of the tropical Salinispora arenicola strain RJA3005, harvested from a PNG marine sediment, produced salinorcinol (3) and salinacetamide (4), which had previously been reported as products of engineered and mutated strains of Amycolatopsis mediterranei, but had not been found before as natural products. An S. arenicola strain RJA4486, harvested from marine sediment collected in the temperate ocean waters off British Columbia, produced the new aminoquinone polyketide salinisporamine (5). Natural products 3, 4, and 5 are putative shunt products of the widely distributed rifamycin biosynthetic pathway.

3,3'-Diindolylmethane Exhibits Significant Metabolism after Oral Dosing in Humans.

3,3'-Diindolylmethane (DIM), a major phytochemical derived from ingestion of cruciferous vegetables, is also a dietary supplement. In preclinical models, DIM is an effective cancer chemopreventive agent and has been studied in a number of clinical trials. Previous pharmacokinetic studies in preclinical and clinical models have not reported DIM metabolites in plasma or urine after oral dosing, and the pharmacological actions of DIM on target tissues is assumed to be solely via the parent compound. Seven subjects (6 males and 1 female) ranging from 26-65 years of age, on a cruciferous vegetable-restricted diet prior to and during the study, took 2 BioResponse DIM 150-mg capsules (45.3 mg DIM/capsule) every evening for one week with a final dose the morning of the first blood draw. A complete time course was performed with plasma and urine collected over 48 hours and analyzed by UPLC-MS/MS. In addition to parent DIM, two monohydroxylated metabolites and 1 dihydroxylated metabolite, along with their sulfate and glucuronide conjugates, were present in both plasma and urine. Results reported here are indicative of significant phase 1 and phase 2 metabolism and differ from previous pharmacokinetic studies in rodents and humans, which reported only parent DIM present after oral administration. 3-((1H-indole-3-yl)methyl)indolin-2-one, identified as one of the monohydroxylated products, exhibited greater potency and efficacy as an aryl hydrocarbon receptor agonist when tested in a xenobiotic response element-luciferase reporter assay using Hepa1 cells. In addition to competitive phytochemical-drug adverse reactions, additional metabolites may exhibit pharmacological activity highlighting the importance of further characterization of DIM metabolism in humans. SIGNIFICANCE STATEMENT: 3,3'-Diindolylmethane (DIM), derived from indole-3-carbinol in cruciferous vegetables, is an effective cancer chemopreventive agent in preclinical models and a popular dietary supplement currently in clinical trials. Pharmacokinetic studies to date have found little or no metabolites of DIM in plasma or urine. In marked contrast, we demonstrate rapid appearance of mono- and dihydroxylated metabolites in human plasma and urine as well as their sulfate and glucuronide conjugates. The 3-((1H-indole-3-yl)methyl)indolin-2-one metabolite exhibited significant aryl hydrocarbon receptor agonist activity, emphasizing the need for further characterization of the pharmacological properties of DIM metabolites.

Geometric Frustration and Long-Range Ordering Induced by Surface Pressure Oscillation in a Langmuir-Blodgett Monolayer of Magnetic Soft Spheres.

As a step toward the bottom-up construction of magnonic systems, this paper demonstrates the use of a large-amplitude surface-pressure annealing technique to generate 2-D order in a Langmuir-Blodgett monolayer of magnetic soft spheres comprising a surfactant-encapsulated polyoxometalate. The films show a distorted square lattice interpreted as due to geometric frustration caused by 2-D confinement between soft walls, one being the air interface and the other the aqueous subphase. Hysteresis and relaxation phenomena in the 2-D layers are suggested to be due to folding and time-dependent interpenetration of surfactant chains.

Synthesis and characterization of mono S-lipidated peptide hydrogels: a platform for the preparation of reactive oxygen species responsive materials.

In this work we report the synthesis of mono lipidated peptides containing a 3-mercaptopropionate linker in the N-terminus by means of a photoinitiated thiol-ene reaction (S-lipidation). We evaluate the self-assembling and hydrogelation properties of a library of mono S-lipidated peptides containing lipid chains of various lengths and demonstrate that hydrogelation was driven by a balance between the lipid chain's hydrophobicity and the peptide's facial hydrophobicity. We further postulate that a simple calculation using estimated values of log D could be used as a predictor of hydrogelation when designing similar systems. A mono S-lipidated peptide containing a short lipid chain that formed hydrogels was fully characterized and a mechanism for the peptide hydrogelation developed. Finally, we demonstrate that the presence of the thioether group in the mono S-lipidated peptide hydrogels, which is a feature lacking in conventional N-acyl lipidated systems, enables the controlled disassembly of the gel via oxidation to the sulfoxide by reactive oxygen species in accordance with a hydrophobicity-modulated strategy. Thus, we conclude that mono S-lipidated peptide hydrogels constitute a novel and simple tool for the development of tissue engineering and targeted drug delivery applications of diseases with overexpression of reactive oxygen species (e.g. degenerative and metabolic diseases, and cancers).

Pulchelloid A, a sesquiterpene lactone from the Canadian prairie plant Gaillardia aristata inhibits mitosis in human cells.

BACKGROUND: The Canadian prairie ecosystem presents a rich source of natural products from plants that are subjected to herbivory by grazing mammals. This type of ecological competition may contribute to the production of natural products of interest in cell biology and medical research. We provide the first biological description of the sesquiterpene lactone, pulchelloid A, which we isolated from the prairie plant, Gaillardia aristata (Asteraceae) and report that it inhibits mitosis in human cells. METHODS AND RESULTS: We found that G. aristata (Blanket flower) extracts were cytotoxic to human cell lines and used phenotypic assays to characterize the bioactivity of extracts. Before dying, cells were characterized by a rounded morphology, phospho-histone H3 signals, mitotic spindles, and active Cdk1. By biology-guided fractionation of Gaillardia extracts, we isolated a sesquiterpene lactone named pulchelloid A. We used immunofluorescence microscopy and observed that cells treated with pulchelloid A have phospho-histone H3 positive chromosomes and a mitotic spindle, confirming that they were in mitosis. Treated cells arrest with an unusual phenotype; they enter a prolonged mitotic arrest in which the spindles become multipolar and the chromosomes acquire histone γH2AX foci, a hallmark of damaged DNA. CONCLUSIONS: We propose that pulchelloid A, a natural product present in the prairie plant Gaillardia aristata, delays cells in mitosis. There is a growing body of evidence that a small number of members of the sesquiterpene lactone chemical family may target proteins that regulate mitosis.

Biologically active marine natural products and their molecular targets discovered using a chemical genetics approach.

Covering: 2000 to 2019The discovery of new natural products that have some combination of unprecedented chemical structures, biological activities of therapeutic interest for urgent medical needs, and new molecular targets provides the fuel that sustains the vitality of natural products chemistry research. Unfortunately, finding these important new compounds is neither routine or trivial and a major challenge is finding effective discovery paradigms. This review presents examples that illustrate the effectiveness of a chemical genetics approach to marine natural product (MNP) discovery that intertwines compound discovery, molecular target identification, and phenotypic response/biological activity. The examples include MNPs that have complex unprecedented structures, new or understudied molecular targets, and potent biological activities of therapeutic interest. A variety of methods to identify molecular targets are also featured.

Aligned Assembly in a 2-D Gel of a Water-Soluble Peptide.

We demonstrate the assembly of a compact, gel-like Langmuir-Blodgett film of rods formed by self-assembly of a ß-sheet-forming water-soluble peptide, Ac-IKHLSVN-NH2, at the surface of aqueous electrolytes. We characterize surface pressure hysteresis and demonstrate shear stiffening of the surface caused by area cycling, which we interpret as due to rearrangement and alignment of the rods. We show strong effects of the electrolyte on the assembly of the elementary rods, which can be related to the Hofmeister series and interpreted by effects on the interaction energies mediated by ions and water. Formation of ß-sheet structures and assembly of these into surface-segregated semicrystalline gels was strongly promoted by ammonium sulfate electrolyte. With ammonium sulfate electrolyte as subphase for Langmuir-Blodgett film deposition, shear stiffening by surface area cycling resulted in very compact films on transfer to a substrate.

Sulforaphane Bioavailability and Chemopreventive Activity in Men Presenting for Biopsy of the Prostate Gland: A Randomized Controlled Trial.

Previous studies suggest compounds such as sulforaphane (SFN) derived from cruciferous vegetables may prevent prostate cancer development and progression. This study evaluated the effect of broccoli sprout extract (BSE) supplementation on blood histone deacetylase (HDAC) activity, prostate RNA gene expression, and tissue biomarkers (histone H3 lysine 18 acetylation (H3K18ac), HDAC3, HDAC6, Ki67, and p21). A total of 98 men scheduled for prostate biopsy were allocated into either BSE (200 µmol daily) or a placebo in our double-blind, randomized controlled trial. We used nonparametric tests to evaluate the differences of blood HDAC activity and prostate tissue immunohistochemistry biomarkers between treatment groups. Further, we performed RNA-Seq analysis on the prostate biopsies and identified 40 differentially expressed genes correlated with BSE treatment, including downregulation of two genes previously implicated in prostate cancer development, AMACR and ARLNC1. Although urine and plasma SFN isothiocyanates and individual SFN metabolites were statistically higher in the treatment group, our results did not show a significant difference in HDAC activity or prostate tissue biomarkers. This study indicates BSE supplementation correlates with changes in gene expression but not with several other prostate cancer biomarkers. More research is required to fully understand the chemopreventive effects of BSE supplementation on prostate cancer.

Directed self-assembly of peptide-diketopyrrolopyrrole conjugates - a platform for bio-organic thin film preparation.

Increased water solubility and long-range intermolecular ordering have been introduced into the fluorescent organic molecule thiophene-diketopyrrolopyrrole (TDPP) via its conjugation to the octapeptide HEFISTAH, which is derived from the protein-protein ß-interface of the homo-tetramer protein diaminopimelate decarboxylase. The octapeptide, and its TDPP mono- and cross-linked conjugates were synthesised using 9-fluorenylmethoxycarbonyl (Fmoc) based solid-phase peptide synthesis (SPPS). Unlike the unmodified peptide, the resulting mono-linked and cross-linked peptides showed a fibrous morphology and formed hydrogels at 4 wt% in water at neutral pH, but failed to assemble at pH 2 and pH 9. Further peptide characterization showed that the TDPP organic core enhances peptide self-assembly and that both peptides assembled into fibers with a parallel ß-sheet structure. Furthermore, UV-vis spectroscopic analysis suggests that the TDPP molecules form H-type aggregates where the chromophores are likely to be co-facially packed, but rotationally and/or laterally offset from one another. This intermolecular coupling indicates that π-π stacking interactions are highly likely - a favourable sign for charge transport. The enhanced aqueous solubility and self-assembling properties of the TDPP-peptide conjugates allowed the successful preparation of thin films. Atomic force microscopy, X-ray diffraction and UV-vis spectroscopic analysis of these thin films revealed that the hybrid materials retained a fibrous morphology, ß-sheet structures and strong intermolecular coupling between neighbouring TDPP molecules. These results open an exciting avenue for bio-organic materials development, through structural and electronic tuning of the TDPP core.

Total synthesis and isolation of citrinalin and cyclopiamine congeners.

Many natural products that contain basic nitrogen atoms--for example alkaloids like morphine and quinine-have the potential to treat a broad range of human diseases. However, the presence of a nitrogen atom in a target molecule can complicate its chemical synthesis because of the basicity of nitrogen atoms and their susceptibility to oxidation. Obtaining such compounds by chemical synthesis can be further complicated by the presence of multiple nitrogen atoms, but it can be done by the selective introduction and removal of functional groups that mitigate basicity. Here we use such a strategy to complete the chemical syntheses of citrinalin B and cyclopiamine B. The chemical connections that have been realized as a result of these syntheses, in addition to the isolation of both 17-hydroxycitrinalin B and citrinalin C (which contains a bicyclo[2.2.2]diazaoctane structural unit) through carbon-13 feeding studies, support the existence of a common bicyclo[2.2.2]diazaoctane-containing biogenetic precursor to these compounds, as has been proposed previously.

Virtual Screening Identifies Chebulagic Acid as an Inhibitor of the M2(S31N) Viral Ion Channel and Influenza A Virus.

The increasing prevalence of drug-resistant influenza viruses emphasizes the need for new antiviral countermeasures. The M2 protein of influenza A is a proton-gated, proton-selective ion channel, which is essential for influenza replication and an established antiviral target. However, all currently circulating influenza A virus strains are now resistant to licensed M2-targeting adamantane drugs, primarily due to the widespread prevalence of an M2 variant encoding a serine to asparagine 31 mutation (S31N). To identify new chemical leads that may target M2(S31N), we performed a virtual screen of molecules from two natural product libraries and identified chebulagic acid as a candidate M2(S31N) inhibitor and influenza antiviral. Chebulagic acid selectively restores growth of M2(S31N)-expressing yeast. Molecular modeling also suggests that chebulagic acid hydrolysis fragments preferentially interact with the highly-conserved histidine residue within the pore of M2(S31N) but not adamantane-sensitive M2(S31). In contrast, chebulagic acid inhibits in vitro influenza A replication regardless of M2 sequence, suggesting that it also acts on other influenza targets. Taken together, results implicate chebulagic acid and/or its hydrolysis fragments as new chemical leads for M2(S31N) and influenza-directed antiviral development.

High-Throughput "FP-Tag" Assay for the Identification of Glycosyltransferase Inhibitors.

Bacterial capsular polysaccharides are important virulence factors. Capsular polysaccharides from several important Gram-negative pathogens share a conserved glycolipid terminus containing 3-deoxy-ß-d- manno-oct-2-ulosonic acid (ß-Kdo). The ß-Kdo glycosyltransferases responsible for synthesis of this conserved glycolipid belong to a new family of glycosyltransferases that shares little homology with other such enzymes, thereby representing an attractive antivirulence target. Here, we report the development of a fluorescence polarization-based, high-throughput screening assay (FP-tag) for ß-Kdo glycosyltransferases, and use it to identify a class of marine natural products as lead inhibitors. This "FP-tag" assay should be readily adaptable to high-throughput screens of other glycosyltransferases.

Toxicokinetics of benzo[a]pyrene in humans: Extensive metabolism as determined by UPLC-accelerator mass spectrometry following oral micro-dosing.

Benzo[a]pyrene (BaP), is a known human carcinogen (International Agency for Research on Cancer (IARC) class 1). The remarkable sensitivity (zepto-attomole 14C in biological samples) of accelerator mass spectrometry (AMS) makes possible, with de minimus risk, pharmacokinetic (PK) analysis following [14C]-BaP micro-dosing of humans. A 46 ng (5 nCi) dose was given thrice to 5 volunteers with minimum 2 weeks between dosing and plasma collected over 72 h. [14C]-BaPeq PK analysis gave plasma Tmax and Cmax values of 1.25 h and 29-82 fg/mL, respectively. PK parameters were assessed by non- compartment and compartment models. Intervals between dosing ranged from 20 to 420 days and had little impact on intra-individual variation. DNA, extracted from peripheral blood mononuclear cells (PBMCs) of 4 volunteers, showed measurable levels (LOD ~ 0.5 adducts/1011 nucleotides) in two individuals 2-3 h post-dose, approximately three orders of magnitude lower than smokers or occupationally-exposed individuals. Little or no DNA binding was detectable at 48-72 h. In volunteers the allelic variants CYP1B1*1/*⁎1, *1/*3 or *3/*3 and GSTM1*0/0 or *1 had no impact on [14C]-BaPeq PK or DNA adduction with this very limited sample. Plasma metabolites over 72 h from two individuals (one CYP1B1*1/*1 and one CYP1B1*3/*3) were analyzed by UPLC-AMS. In both individuals, parent [14C]-BaP was a minor constituent even at the earliest time points and metabolite profiles markedly distinct. AMS, coupled with UPLC, could be used in humans to enhance the accuracy of pharmacokinetics, toxicokinetics and risk assessment of environmental carcinogens.

Assembly of Protein Stacks With in Situ Synthesized Nanoparticle Cargo.

The ability of proteins to form hierarchical structures through self-assembly provides an opportunity to synthesize and organize nanoparticles. Ordered nanoparticle assemblies are a subject of widespread interest due to the potential to harness their emergent functions. In this work, the toroidal-shaped form of the protein peroxiredoxin, which has a pore size of 7 nm, was used to organize iron oxyhydroxide nanoparticles. Iron in the form of Fe2+ was sequestered into the central cavity of the toroid ring using metal-binding sites engineered there and then hydrolyzed to form iron oxyhydroxide particles bound into the protein pore. By precise manipulation of the pH, the mineralized toroids were organized into stacks confining one-dimensional nanoparticle assemblies. We report the formation and the procedures leading to the formation of such nanostructures and their characterization by chromatography and microscopy. Electrostatic force microscopy clearly revealed the formation of iron-containing nanorods as a result of the self-assembly of the iron-loaded protein. This research bodes well for the use of peroxiredoxin as a template with which to form nanowires and structures for electronic and magnetic applications.

Serpulanines A to C, N-Oxidized Tyrosine Derivatives Isolated from the Sri Lankan Fungus Serpula sp.: Structure Elucidation, Synthesis, and Histone Deacetylase Inhibition.

Serpulanines A (1), B (2), and C (3) have been isolated from extracts of the rare Sri Lankan macrofungus Serpula sp. The structures of 1, 2, and 3 were elucidated by a combination of spectroscopic and single-crystal X-ray diffraction analyses. Serpulanines A (1) and B (2) both contain the rare (E)-2-hydroxyimino hydroxamic acid functional group array. A proposed biogenesis for serpulanine B (2) suggests that its (E)-2-hydroxyimino hydroxamic acid moiety arises from a diketopiperazine precursor. Synthetic serpulanine A (1) inhibited class I/II histone deacetylases in murine metastatic lung carcinoma cells with an IC50 of 7 µM.