Characterization of the active site in the thiocyanate-forming protein from Thlaspi arvense (TaTFP) using EPR spectroscopy.

Glucosinolates are plant thioglucosides, which act as chemical defenses. Upon tissue damage, their myrosinase-catalyzed hydrolysis yields aglucones that rearrange to toxic isothiocyanates. Specifier proteins such as thiocyanate-forming protein from Thlaspi arvense (TaTFP) are non-heme iron proteins, which capture the aglucone to form alternative products, e.g. nitriles or thiocyanates. To resolve the electronic state of the bound iron cofactor in TaTFP, we applied continuous wave electron paramagnetic resonance (CW EPR) spectroscopy at X-and Q-band frequencies (∼9.4 and ∼34 GHz). We found characteristic features of high spin and low spin states of a d 5 electronic configuration and local rhombic symmetry during catalysis. We monitored the oxidation states of bound iron during conversion of allylglucosinolate by myrosinase and TaTFP in presence and absence of supplemented Fe2+. Without added Fe2+, most high spin features of bound Fe3+ were preserved, while different g'-values of the low spin part indicated slight rearrangements in the coordination sphere and/or structural geometry. We also examined involvement of the redox pair Fe3+/Fe2 in samples with supplemented Fe2+. The absence of any EPR signal related to Fe3+ or Fe2+ using an iron-binding deficient TaTFP variant allowed us to conclude that recorded EPR signals originated from the bound iron cofactor.

Bichromonol, a dimeric coumarin with anti-HIV activity from the stem bark of Hypericum roeperianum.

Infectious diseases caused by viruses like HIV and SARS-COV-2 (COVID-19) pose serious public health threats. In search for new antiviral small molecules from chemically underexplored Hypericum species, a previously undescribed atropisomeric C8-C8' linked dimeric coumarin named bichromonol (1) was isolated from the stem bark of Hypericum roeperianum. The structure was elucidated by MS data and NMR spectroscopy. The absolute configuration at the biaryl axis was determined by comparing the experimental ECD spectrum with those calculated for the respective atropisomers. Bichromonol was tested in cell-based assays for cytotoxicity against MT-4 (CC50 = 54 µM) cells and anti-HIV activity in infected MT-4 cells. It exhibits significant activity at EC50 = 6.6-12.0 µM against HIV-1 wild type and its clinically relevant mutant strains. Especially, against the resistant variants A17 and EFVR, bichromonol is more effective than the commercial drug nevirapine and might thus have potential to serve as a new anti-HIV lead.

A single cytochrome P450 oxidase from Solanum habrochaites sequentially oxidizes 7-epi-zingiberene to derivatives toxic to whiteflies and various microorganisms.

Secretions from glandular trichomes potentially protect plants against a variety of aggressors. In the tomato clade of the Solanum genus, glandular trichomes of wild species produce a rich source of chemical diversity at the leaf surface. Previously, 7-epi-zingiberene produced in several accessions of Solanum habrochaites was found to confer resistance to whiteflies (Bemisia tabaci) and other insect pests. Here, we report the identification and characterisation of 9-hydroxy-zingiberene (9HZ) and 9-hydroxy-10,11-epoxyzingiberene (9H10epoZ), two derivatives of 7-epi-zingiberene produced in glandular trichomes of S. habrochaites LA2167. Using a combination of transcriptomics and genetics, we identified a gene coding for a cytochrome P450 oxygenase, ShCYP71D184, that is highly expressed in trichomes and co-segregates with the presence of the zingiberene derivatives. Transient expression assays in Nicotiana benthamiana showed that ShCYP71D184 carries out two successive oxidations to generate 9HZ and 9H10epoZ. Bioactivity assays showed that 9-hydroxy-10,11-epoxyzingiberene in particular exhibits substantial toxicity against B. tabaci and various microorganisms including Phytophthora infestans and Botrytis cinerea. Our work shows that trichome secretions from wild tomato species can provide protection against a wide variety of organisms. In addition, the availability of the genes encoding the enzymes for the pathway of 7-epi-zingiberene derivatives makes it possible to introduce this trait in cultivated tomato by precision breeding.

Natural Polyketides Isolated from the Endophytic Fungus Phomopsis sp. CAM212 with a Semisynthetic Derivative Downregulating the ERK/IκBα Signaling Pathways.

Three previously undescribed natural products, phomopsinin A - C (1:  - 3: ), together with three known compounds, namely, cis-hydroxymellein (4: ), phomoxanthone A (5: ) and cytochalasin L-696,474 (6: ), were isolated from the solid culture of Phomopsis sp. CAM212, an endophytic fungus obtained from Garcinia xanthochymus. Their structures were determined on the basis of spectroscopic data, including IR, NMR, and MS. The absolute configurations of 1: and 2: were assigned by comparing their experimental and calculated ECD spectra. Acetylation of compound 1: yielded 1A: , a new natural product derivative that was tested together with other isolated compounds on lipopolysaccharide-stimulated RAW 264.7 cells. Cytochalasin L-696,474 (6: ) was found to significantly inhibit nitric oxide production, but was highly cytotoxic to the treated cells, whereas compound 1: slightly inhibited nitric oxide production, which was not significantly different compared to lipopolysaccharide-treated cells. Remarkably, the acetylated derivative of 1: , compound 1A: , significantly inhibited nitric oxide production with an IC50 value of 14.8 µM and no cytotoxic effect on treated cells, thereby showing the importance of the acetyl group in the anti-inflammatory activity of 1A: . The study of the mechanism of action revealed that 1A: decreases the expression of inducible nitric oxide synthase, cyclooxygenase 2, and proinflammatory cytokine IL-6 without an effect on IL-1ß expression. Moreover, it was found that 1A: exerts its anti-inflammatory activity in lipopolysaccharide-stimulated RAW 264.7 macrophage cells by downregulating the activation of ERK1/2 and by preventing the translocation of nuclear factor κB. Thus, derivatives of phomopsinin A (1: ), such as compound 1A: , could provide new anti-inflammatory leads.

Expanding the Diversity of Plant Monoterpenoid Indole Alkaloids Employing Human Cytochrome P450 3A4.

Human drug-metabolizing cytochrome P450 monooxygenases (CYPs) have enormous substrate promiscuity; this makes them promising tools for the expansion of natural product diversity. Here, we used CYP3A4 for the targeted diversification of a plant biosynthetic route leading to monoterpenoid indole alkaloids. In silico, in vitro and in planta studies proved that CYP3A4 was able to convert the indole alkaloid vinorine into vomilenine, the former being one of the central intermediates in the ajmaline pathway in the medicinal plant Rauvolfia serpentina (L.) Benth. ex Kurz. However, to a much larger extent, the investigated conversion yielded vinorine (19R,20R)-epoxide, a new metabolite with an epoxide functional group that is rare for indole alkaloids. The described work represents a successful example of combinatorial biosynthesis towards an increase in biodiversity of natural metabolites. Moreover, characterisation of the products of the in vitro and in planta transformation of potential pharmaceuticals with human CYPs might be indicative of the route of their conversion in the human organism.

Insights into the Phytochemistry of the Cuban Endemic Medicinal Plant Phyllanthus orbicularis: Fideloside, a Novel Bioactive 8-C-glycosyl 2,3-Dihydroflavonol.

Phyllanthus orbicularis (Phyllanthaceae) is an endemic evergreen tropical plant of Cuba that grows in the western part of the island and is used in traditional medicine as an infusion. The aqueous extract of this plant presents a wide range of pharmacological activitiessuch as antimutagenic, antioxidant and antiviral effects. Given the many beneficial effects and the great interest in the development of new pharmacological products from natural sources, the aim of this work was to investigate the phytochemistry of this species and to elucidate the structure of the main bioactive principles. Besides the presence of several known polyphenols, the major constituent was hitherto not described. The chemical structure of this compound, here named Fideloside, was elucidated by means of HR-ESIMS/MSn, 1D/2D NMR, FT-IR, and ECD as (2R,3R)-(-)-3',4',5,7-tetrahydroxydihydroflavonol-8-C-ß-D-glucopyranoside. The compound, as well as the plant aqueous preparations, showed promising bioactive properties, i.e., anti-inflammatory capacity in human explanted monocytes, corroborating future pharmacological use for this new natural C-glycosyl flavanonol.

Tricyclic Acylphloroglucinols from Hypericum lanceolatum and Regioselective Synthesis of Selancins A and B.

The chemical investigation of the chloroform extract of Hypericum lanceolatum guided by (1)H NMR, ESIMS, and TLC profiles led to the isolation of 11 new tricyclic acylphloroglucinol derivatives, named selancins A-I (1-9) and hyperselancins A and B (10 and 11), along with the known compound 3-O-geranylemodin (12), which is described for a Hypericum species for the first time. Compounds 8 and 9 are the first examples of natural products with a 6-acyl-2,2-dimethylchroman-4-one core fused with a dimethylpyran unit. The new compounds 1-9 are rare acylphloroglucinol derivatives with two fused dimethylpyran units. Compounds 10 and 11 are derivatives of polycyclic polyprenylated acylphloroglucinols related to hyperforin, the active component of St. John's wort. Their structures were elucidated by UV, IR, extensive 1D and 2D NMR experiments, HRESIMS, and comparison with the literature data. The absolute configurations of 5, 8, 10, and 11 were determined by comparing experimental and calculated electronic circular dichroism spectra. Compounds 1 and 2 were synthesized regioselectively in two steps. The cytotoxicity of the crude extract (88% growth inhibition at 50 µg/mL) and of compounds 1-6, 8, 9, and 12 (no significant growth inhibition up to a concentration of 10 mM) against colon (HT-29) and prostate (PC-3) cancer cell lines was determined. No anthelmintic activity was observed for the crude extract.

Structure-Activity Relationships of Antimicrobial Gallic Acid Derivatives from Pomegranate and Acacia Fruit Extracts against Potato Bacterial Wilt Pathogen.

Bacterial wilts of potato, tomato, pepper, and or eggplant caused by Ralstonia solanacearum are among the most serious plant diseases worldwide. In this study, the issue of developing bactericidal agents from natural sources against R. solanacearum derived from plant extracts was addressed. Extracts prepared from 25 plant species with antiseptic relevance in Egyptian folk medicine were screened for their antimicrobial properties against the potato pathogen R. solancearum by using the disc-zone inhibition assay and microtitre plate dilution method. Plants exhibiting notable antimicrobial activities against the tested pathogen include extracts from Acacia arabica and Punica granatum. Bioactivity-guided fractionation of A. arabica and P. granatum resulted in the isolation of bioactive compounds 3,5-dihydroxy-4-methoxybenzoic acid and gallic acid, in addition to epicatechin. All isolates displayed significant antimicrobial activities against R. solanacearum (MIC values 0.5-9 mg/ml), with 3,5-dihydroxy-4-methoxybenzoic acid being the most effective one with a MIC value of 0.47 mg/ml. We further performed a structure-activity relationship (SAR) study for the inhibition of R. solanacearum growth by ten natural, structurally related benzoic acids.

Screening of synthetic and natural product databases: Identification of novel androgens and antiandrogens.

The androgen receptor is an important pharmaceutical target for a variety of diseases. This paper presents an in silico/in vitro screening procedure to identify new androgen receptor ligands. The two-step virtual screening procedure uses a three-dimensional pharmacophore model and a docking/scoring routine. About 39,000 filtered compounds were docked with PLANTS and scored by Chemplp. Subsequent to virtual screening, 94 compounds, including 28 steroidal and 66 nonsteroidal compounds, were tested by an androgen receptor fluorescence polarization ligand displacement assay. As a result, 30 compounds were identified that show a relative binding affinity of more than 50% in comparison to 100 nM dihydrotestosterone and were classified as androgen receptor binders. For 11 androgen receptor binders of interest IC50 and Ki values were determined. The compound with the highest affinity exhibits a Ki value of 10.8 nM. Subsequent testing of the 11 compounds in a PC-3 and LNCaP multi readout proliferation assay provides insights into the potential mode of action. Further steroid receptor ligand displacement assays and docking studies on estrogen receptors α and ß, glucocorticoid receptor, and progesterone receptor gave information about the specificity of the 11 most active compounds.

Inhibition of human intestinal α-glucosidases by calystegines.

Calystegines are polyhydroxylated nortropane alkaloids found in Convolvulaceae, Solanaceae, and other plant families. These plants produce common fruits and vegetables. The calystegine structures resemble sugars and suggest interaction with enzymes of carbohydrate metabolism. Maltase and sucrase are α-glucosidases contributing to human carbohydrate degradation in the small intestine. Inhibition of these enzymes by orally administered drugs is one option for treatment of diabetes mellitus type 2. In this study, inhibition of maltase and sucrase by calystegines A3 and B2 purified from potatoes was investigated. In silico docking studies confirmed binding of both calystegines to the active sites of the enzymes. Calystegine A3 showed low in vitro enzyme inhibition; calystegine B2 inhibited mainly sucrose activity. Both compounds were not transported by Caco-2 cells indicating low systemic availability. Vegetables rich in calystegine B2 should be further investigated as possible components of a diet preventing a steep increase in blood glucose after a carbohydrate-rich meal.

Diallylpolysulfides induce growth arrest and apoptosis.

Garlic-derived organo sulphur compounds such as diallylsulfides provide a significant protection against carcinogenesis. Chemically synthesized, and highly pure diallylsulfides with a chain of 1-4 sulphur atoms, as well as a range of control compounds, were employed to investigate the influence of these agents on cell viability, cell cycle arrest and induction of apoptosis in HCT116 human colon cancer cells. Diallyltrisulfide, and even more efficiently diallyltetrasulfide treatment of HCT116 cells led to a reduced cell viability, cell cycle arrest and apoptosis. A similar activity was found for the propyl-analogues, while mono- and disulfides were considerably less active. Initial calculations point toward the ability of tri- and tetrasulfides to form reactive oxygen species (ROS). Here, we found that the induction of apoptosis was indeed dependent on the redox-state of the cell, with anti-oxidants being able to prevent sulfide-induced apoptosis. Furthermore, using HCT116 cells which were either positive or negative for p53 revealed that p53 is clearly dispensable for induction of apoptosis. Growth arrest and induction of apoptosis is associated with a considerable reduction of the level of cdc25C. These results support the therapeutic potential of polysulfides and allow insight into the mechanisms based on the polysulfide biochemistry.

Protein structure modeling indicates hexahistidine-tag interference with enzyme activity.

Unusual kinetic characteristics of tropinone reductase, an enzyme in the family of short chain dehydrogenases, prompted to investigate a possible impact of the hexahistidine affinity tag on catalytic properties. Comparison of enzymes from Solanum dulcamara, Solanaceae, tagged at the N-terminus or at the C-terminus revealed that the C-terminally tagged form was functionally impaired. Protein modeling indicated that the hexahistidine tag attached at the C-terminus but not at the N-terminus of the polypeptide can interfere with the active site by steric or electrostatic interactions. In consequence, protein modeling is suggested before enzyme expression with affinity tags to estimate possible interactions of affinity tags with the active center.

Selenium in chemistry and biochemistry in comparison to sulfur.

What makes selenoenzymes--seen from a chemist's view--so special that they cannot be substituted by just more analogous or adapted sulfur proteins? This review compiles and compares physicochemical properties of selenium and sulfur, synthetic routes to selenocysteine (Sec) and its peptides, and comparative studies of relevant thiols and selenols and their (mixed) dichalcogens, required to understand the special role of selenium in selenoproteins on the atomic molecular level. The biochemically most relevant differences are the higher polarizability of Se- and the lower pKa of SeH. The latter has a strikingly different pH-dependence than thiols, with selenols being active at much lower pH. Finally, selected typical enzymatic mechanisms which involve selenocysteine are critically discussed, also in view of the authors' own results.

Influence of pH and flanking serine on the redox potential of S-S and S-Se bridges of Cys-Cys and Cys-Sec peptides.

In selenocysteine (Sec, U)-containing proteins the selenenylsulfide bridge and its reduced thiol-selenol counterpart are usually the significant species. An important role for serine as flanking amino acid in the redox potential of S-S and S-Se bridges was proposed for some thioredoxin reductases. To check the generality of this proposal, model tetrapeptides (GCCG, SCCG, GCCS, SCCS, GCUG, SCUG, GCUS, SCUS) were synthesized, including the GCUG sequence of human thioredoxin reductase. The influence on the redox potential of S-Se and S-S bridges as a function of pH and of serine at different positions reveals (i) a strong general pH dependence, and (ii) a significant influence of flanking serine on disulfide only at basic pH.