Intrinsic disorder and conformational coexistence in auxin coreceptors.

AUXIN/INDOLE 3-ACETIC ACID (Aux/IAA) transcriptional repressor proteins and the TRANSPORT INHIBITOR RESISTANT 1/AUXIN SIGNALING F-BOX (TIR1/AFB) proteins to which they bind act as auxin coreceptors. While the structure of TIR1 has been solved, structural characterization of the regions of the Aux/IAA protein responsible for auxin perception has been complicated by their predicted disorder. Here, we use NMR, CD and molecular dynamics simulation to investigate the N-terminal domains of the Aux/IAA protein IAA17/AXR3. We show that despite the conformational flexibility of the region, a critical W-P bond in the core of the Aux/IAA degron motif occurs at a strikingly high (1:1) ratio of cis to trans isomers, consistent with the requirement of the cis conformer for the formation of the fully-docked receptor complex. We show that the N-terminal half of AXR3 is a mixture of multiple transiently structured conformations with a propensity for two predominant and distinct conformational subpopulations within the overall ensemble. These two states were modeled together with the C-terminal PB1 domain to provide the first complete simulation of an Aux/IAA. Using MD to recreate the assembly of each complex in the presence of auxin, both structural arrangements were shown to engage with the TIR1 receptor, and contact maps from the simulations match closely observations of NMR signal-decreases. Together, our results and approach provide a platform for exploring the functional significance of variation in the Aux/IAA coreceptor family and for understanding the role of intrinsic disorder in auxin signal transduction and other signaling systems.

Non-specific effects of the CINNAMATE-4-HYDROXYLASE inhibitor piperonylic acid.

Chemical inhibitors are often implemented for the functional characterization of genes to overcome the limitations associated with genetic approaches. Although it is well established that the specificity of the compound is key to success of a pharmacological approach, off-target effects are often overlooked or simply neglected in a complex biological setting. Here we illustrate the cause and implications of such secondary effects by focusing on piperonylic acid (PA), an inhibitor of CINNAMATE-4-HYDROXYLASE (C4H) that is frequently used to investigate the involvement of lignin during plant growth and development. When supplied to plants, we found that PA is recognized as a substrate by GRETCHEN HAGEN 3.6 (GH3.6), an amido synthetase involved in the formation of the indole-3-acetic acid (IAA) conjugate IAA-Asp. By competing for the same enzyme, PA interferes with IAA conjugation, resulting in an increase in IAA concentrations in the plant. In line with the broad substrate specificity of the GH3 family of enzymes, treatment with PA increased not only IAA levels but also those of other GH3-conjugated phytohormones, namely jasmonic acid and salicylic acid. Finally, we found that interference with the endogenous function of GH3s potentially contributes to phenotypes previously observed upon PA treatment. We conclude that deregulation of phytohormone homeostasis by surrogate occupation of the conjugation machinery in the plant is likely a general phenomenon when using chemical inhibitors. Our results hereby provide a novel and important basis for future reference in studies using chemical inhibitors.

Impact of Melatonin Deficit on Emotional Status and Oxidative Stress-Induced Changes in Sphingomyelin and Cholesterol Level in Young Adult, Mature, and Aged Rats.

The pineal gland regulates the aging process via the hormone melatonin. The present report aims to evaluate the effect of pinealectomy (pin) on behavioral and oxidative stress-induced alterations in cholesterol and sphingomyelin (SM) levels in young adult, mature and aging rats. Sham and pin rats aged 3, 14 and 18 months were tested in behavioral tests for motor activity, anxiety, and depression. The ELISA test explored oxidative stress parameters and SM in the hippocampus, while total cholesterol was measured in serum via a commercial autoanalyzer. Mature and aged sham rats showed low motor activity and increased anxiety compared to the youngest rats. Pinealectomy affected emotional responses, induced depressive-like behavior, and elevated cholesterol levels in the youngest rats. However, removal of the pineal gland enhanced oxidative stress by diminishing antioxidant capacity and increasing the MDA level, and decreased SM level in the hippocampus of 14-month-old rats. Our findings suggest that young adult rats are vulnerable to emotional disturbance and changes in cholesterol levels resulting from melatonin deficiency. In contrast, mature rats with pinealectomy are exposed to an oxidative stress-induced decrease in SM levels in the hippocampus.

The Role of Melatonin on Behavioral Changes and Concomitant Oxidative Stress in icvAß1-42 Rat Model with Pinealectomy.

One of the pathological hallmarks of Alzheimer's disease (AD) associated with its progression that contributes to ß-amyloid (Aß) generation is oxidative stress (OS). Clinical data suggest that melatonin is a potent antioxidant that might be effective in the adjunctive therapy of this neurodegenerative disease. The present study aimed to explore the role of melatonin on behavioral changes and markers of OS in three rat models, namely, pinealectomy (pin) model of melatonin deficit, intracerebroventricular (icv)Aß1-42 model of AD, and combination of both pin and Aß1-42 model (pin+icvAß1-42). The chronic injection with vehicle/melatonin (50 mg/kg, i.p. for 40 days) started on the same day of sham/pin and icv vehicle/Aß1-42 infusion procedures. Anxiety in the open field and the elevated plus-maze test and cognitive responses in the object recognition test were tested between the 30th-35th day after the surgical procedures. Markers of OS in the frontal cortex (FC) and hippocampus were detected by the ELISA method. Melatonin treatment corrected the exacerbated anxiety response only in the pin+icvAß1-42 model while it alleviated the cognitive impairment in the three models. Pinealectomy disturbed the antioxidant system via enhanced SOD activity and decreased GSH levels both in the FC and hippocampus. The Aß1-42 model decreased the SOD activity in the FC and elevated the MDA level in the two brain structures. The pin+icvAß1-42 model impaired the antioxidant system and elevated lipid peroxidation. Melatonin supplementation restored only the elevated MDA level of icvAß1-42 and pin+icvAß1-42 model in the hippocampus. In conclusion, our study reveals that the pin+icvAß1-42 rat model triggers more pronounced anxiety and alterations in markers of OS that may be associated with melatonin deficit concomitant to icvAß1-42-induced AD pathology.

Evaluation of neurobiological and antioxidant effects of novel melatonin analogs in mice.

Based on the pharmacophore model of melatonin (MT1) receptor, we recently synthesized a series of indole derivatives that showed anticonvulsant activity with low neurotoxicity and hepatotoxicity in rodents. In the present study, the three most potent C3-modified derivatives with hydrazine structure 3c, 3e, and 3f, with 2-chlorophenyl, 2-furyl, and 2-thienyl fragments, respectively, were selected, and their neurobiological activity was explored in mice. In Experiment #1, the dose-dependent anxiolytic effect of a single i.p. administration of the novel compounds at doses of 10, 30, and 60 mg/kg were studied in the open field (OF) test. In Experiment#2, the analgesic effect of 3c, 3e, and 3f (30-100 mg/kg) was tested in the hot plate test and formalin test. Experiment#3 was designed to assess the antidepressant-like activity of 3c, 3e, and 3f (10-60 mg/kg). The forced swimming test (FST) and tail suspension test (TST)-induced effect on markers of oxidative stress in the frontal cortex (FC), and the hippocampus was evaluated. Melatonin was used in the same doses as melatonin analogs in all three experiments as a positive control. Desipramine (10 mg/kg) was also applied as a control in the FST. The three melatonin analogs bearing hydrazide/hydrazone substitution at 3C of the indol scaffold demonstrated improved antidepressant-like activity compared to the melatonin. The tested substances are devoided of anxiolytic effects. The antioxidant activity of the melatonin analogs and analgesic potential is comparable to that of melatonin. The 3C substitution with hydrazide/hydrazone moiety substantially contributes to the antidepressant and antioxidant activity of the melatonin analogs.

Single-Chain Glycopolymer Folding via Host-Guest Interactions and Its Unprecedented Effect on DC-SIGN Binding.

Reversible self-folding actions of natural biomacromolecules play crucial roles for specific and unique biological functions in Nature. Hence, controlled folding of single polymer chains has attracted significant attention in recent years. Herein, reversible single-chain folded glycopolymer structures in α-shape with different density of sugar moieties in the knot were created. The influence of folding as well as the sugar density in the knot was investigated on the binding capability with lectins, such as ConA, DC-SIGN, and DC-SIGNR. The synthesis of triblock glycocopolymers bearing ß-CD and adamantane for the host-guest interaction and also mannose residues for the lectin interaction was achieved using the reversible addition-fragmentation chain transfer (RAFT) polymerization technique. The reversible single-chain folding of glycopolymers was achieved under a high dilution of an aqueous solution and the self-assembled folding was monitored by 2D nuclear overhauser enhancement spectroscopy (NOESY) NMR and dynamic light scattering. The lectin binding profiles consistently provided an unprecedented effect of single chain folding as the single-chain folded structures enhanced greatly the binding ability in comparison to the unfolded linear structures.

Block-Sequence-Specific Glycopolypeptides with Selective Lectin Binding Properties.

Glycopolypeptides with defined block sequences were prepared by sequential addition of two different N-carboxyanhydrides (NCAs), followed by selective deprotection and functionalization of predefined positions within the polypeptide backbone. The sequential arrangement of the galactose units and the block-sequence length have been systematically varied. All the glycopolypeptides have been obtained with a similar overall composition and comparable molecular weights. Circular dichroism measurements revealed some dependence of the secondary structure on the primary composition of the glycopolypeptides at physiological pH. While statistical, diblock, and tetrablock glycopolypeptides adopted a random coil conformation, the octablock glycopolypeptide was mostly α-helical. The ability to selectively bind to lectins was investigated by turbidity measurements as well as surface plasmon resonance (SPR) studies. It was found that the extent of binding was dependent on the position of the galactose units and thus the primary glycopolypeptide structure. The octablock glycopolypeptide favored interaction with lectin RCA120 while the tetrablock glycopolypeptide demonstrated the strongest binding activity to Galectin-3. The results suggest that different lectins are very sensitive to glyco coding and that precise control of carbohydrate units in synthetic polymeric glycopeptides will remain important.

Characterization of DNA-conjugated compounds using a regenerable chip.

DNA-encoded chemical library (DECL) technology has emerged as a new avenue in the field of drug discovery. Combined with high-throughput sequencing, DECL selection experiments can provide not only many lead compounds but also insights into the structure-affinity relationship. However, the counts of individual DNA codes reflect, but cannot be used to precisely rank, the binding affinities of the corresponding compounds to protein targets. Herein, we describe a chip-based approach to realize an automated high-throughput assay for the kinetic characterization of the interaction between DNA-conjugated small organic compounds and protein targets. Importantly, this method can be applied to both single-pharmacophore DECLs and self-assembled dual-pharmacophore DECLs.

Amphipathic DNA origami nanoparticles to scaffold and deform lipid membrane vesicles.

We report a synthetic biology-inspired approach for the engineering of amphipathic DNA origami structures as membrane-scaffolding tools. The structures have a flat membrane-binding interface decorated with cholesterol-derived anchors. Sticky oligonucleotide overhangs on their side facets enable lateral interactions leading to the formation of ordered arrays on the membrane. Such a tight and regular arrangement makes our DNA origami capable of deforming free-standing lipid membranes, mimicking the biological activity of coat-forming proteins, for example, from the I-/F-BAR family.

A Standardized Extract of Petasites hybridus L., Containing the Active Ingredients Petasins, Acts as a Pro-Oxidant and Triggers Apoptosis through Elevating of NF-κB in a Highly Invasive Human Breast Cancer Cell Line.

BACKGROUND: Common butterbur (Petasites hybridus L.) is a traditional medicinal plant with numerous therapeutic properties among which is its recently uncovered anti-tumor activity. The present study aims to examine the activity of a standardized Bulgarian Petasites hybridus L. root extract, containing the active ingredients petasins, on the human breast cancer cell line MDA-MB-231 and non-cancerous MCF-10A cells. Specifically, we examined cell death, oxidative stress, and nuclear factor kappa-B (NF-κB) signaling. METHODS: A standardized butterbur powdered extract containing a minimum of 15% petasins was used. A lipophilic extract was obtained from subterranean portion of the plant of Bulgarian populations of Petasites hybridus using liquid-liquid extraction after completely removing pyrrolizidine alkaloids. The induction of apoptosis and necrosis was analyzed by flow cytometry, and oxidative stress biomarkers and NF-κB were measured using enzyme-linked immunosorbent assay (ELISA). RESULTS: Petasites hybridus L. root extract triggered apoptosis in a cancer-specific fashion and induced a moderate oxidative stress characterized by diminished glutathione (GSH) levels and elevated malondialdehyde (MDA) levels in MDA-MB-231 72 h after treatment. NF-κB levels were higher in cancer cells after treatment with IC50 and IC75 doses, this suggested that the NF-κB pathway was activated in response to oxidative stress leading to the induction of apoptosis. MCF-10A cells were affected to a lesser extent by the Petasites hybridus extract, and the adaptive response of their antioxidant defense system halted oxidative stress. CONCLUSIONS: Overall, these results indicate that Petasites hybridus L. root extract selectively acts as a pro-oxidant in breast cancer cells and thus represents a potential therapeutic option for cancer treatment with fewer side effects.

The differential binding and biological efficacy of auxin herbicides.

BACKGROUND: Auxin herbicides have been used for selective weed control for 75 years and they continue to be amongst the most widely used weed control agents globally. The auxin herbicides fall into five chemical classes, with two herbicides not classified, and in all cases it is anticipated that recognition in the plant starts with binding to the Transport Inhibitor Response 1 (TIR1) family of auxin receptors. There is evidence that some classes of auxins act selectively with certain clades of receptors, although a comprehensive structure-activity relationship has not been available. RESULTS: Using purified receptor proteins to measure binding efficacy we have conducted quantitative structure activity relationship (qSAR) assays using representative members of the three receptor clades in Arabidopsis, TIR1, AFB2 and AFB5. Complementary qSAR data for biological efficacy at the whole-plant level using root growth inhibition and foliar phytotoxicity assays have also been analyzed for each family of auxin herbicides, including for the afb5-1 receptor mutant line. CONCLUSIONS: Comparisons of all these assays highlight differences in receptor selectivity and some systematic differences between results for binding in vitro and activity in vivo. The results could provide insights into weed spectrum differences between the different classes of auxin herbicides, as well as the potential resistance and cross-resistance implications for this herbicide class. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

1H-benzimidazole-2-yl hydrazones as tubulin-targeting agents: Synthesis, structural characterization, anthelmintic activity and antiproliferative activity against MCF-7 breast carcinoma cells and molecular docking studies.

In the present study, fifteen benzimidazolyl-2-hydrazones 7a-7o of fluoro-, hydroxy- and methoxy-substituted benzaldehydes and 1,3-benzodioxole-5-carbaldehyde were synthesized and their structure was identified by IR, NMR, and elemental analysis. The compounds 7j 2-(3-hydroxybenzylidene)-1-(5(6)-methyl-1H-benzimidazol-2-yl)hydrazone and 7i 2-(3-hydroxybenzylidene)-1-(1H-benzimidazol-2-yl)hydrazone have exerted the strongest anthelmintic activity (100% after 24 h incubation period at 37 °C) against isolated muscle larvae of Trichinella spiralis in an in vitro experiment. The in vitro cytotoxicity assay towards MCF-7 breast cancer cells and mouse embryo fibroblasts 3T3 showed that the studied benzimidazolyl-2-hydrazones exhibit low to moderate cytotoxic effects. The ability of the studied benzimidazolyl-2-hydrazones to modulate microtubule polymerization was confirmed and suggested that their anthelmintic action is mediated through inhibition of the tubulin polymerization likewise the other known benzimidazole anthelmitics. It was also shown that the four most promising benzimidazolyl-2-hydrazones do not affect significantly the AChE activity even at high tested concentration, thus indicating that they do not have the potential for neurotoxic effects. The binding mode of compounds 7j and 7n in the colchicine-binding site of tubulin were clarified by molecular docking simulations. Taken together, these results demonstrate that for the synthesized benzimidazole derivatives the anthelmintic activity against T. spiralis and the inhibition of tubulin polymerization are closely related.

Strengthening CoViD-19 therapy via combinations of RAS modulators.

Evidence has accumulated that the pathology of CoViD-19 is strongly related to the renin-angiotensin system (RAS). The blockage of the angiotensin converting enzyme 2 (ACE2) by the SARS-CoV-2 virus leads to downstream consequences such as increased vascular tone, extensive fibrosis and pronounced immune reactions. Different approaches to tackle the adverse viral effects by compensating the lost ACE2 function have been suggested. Here, we use an unequal-arm lever model to describe a simplified version of the biased regulation exercised by the angiotensin II and angiotensin-(1-7) hormones, which are the substrate and the product of ACE2, respectively. We reason upon the lever dynamics and its disruptions caused by the virus, and propose that a combination of RAS modulators will most efficiently compensate the imbalance due to the excess of angiotensin II and the scarcity of angiotensin-(1-7). Specifically, we focus on the possible benefits of the simultaneous application of two agents, a MAS-receptor agonist and an angiotensin-II-type-2-receptor agonist. We conjecture that this combination has the potential to introduce a beneficial synergistic action that promotes anti-hypoxic, anti-fibrotic and anti-proliferative effects, thereby improving the clinical management of acute and chronic CoViD-19 pathologies.

Hyaluronan (HA)-inspired glycopolymers as molecular tools for studying HA functions.

Hyaluronic acid (HA), the only non-sulphated glycosaminoglycan, serves numerous structural and biological functions in the human body, from providing viscoelasticity in tissues to creating hydrated environments for cell migration and proliferation. HA is also involved in the regulation of morphogenesis, inflammation and tumorigenesis through interactions with specific HA-binding proteins. Whilst the physicochemical and biological properties of HA have been widely studied for decades, the exact mechanisms by which HA exerts its multiple functions are not completely understood. Glycopolymers offer a simple and precise synthetic platform for the preparation of glycan analogues, being an alternative to the demanding synthetic chemical glycosylation. A library of homo, statistical and alternating HA glycopolymers were synthesised by reversible addition-fragmentation chain transfer polymerisation and post-modification utilising copper alkyne-azide cycloaddition to graft orthogonal pendant HA monosaccharides (N-acetyl glucosamine: GlcNAc and glucuronic acid: GlcA) onto the polymer. Using surface plasmon resonance, the binding of the glycopolymers to known HA-binding peptides and proteins (CD44, hyaluronidase) was assessed and compared to carbohydrate-binding proteins (lectins). These studies revealed potential structure-binding relationships between HA monosaccharides and HA receptors and novel HA binders, such as Dectin-1 and DEC-205 lectins. The inhibitory effect of HA glycopolymers on hyaluronidase (HAase) activity was also investigated suggesting GlcNAc- and GlcA-based glycopolymers as potential HAase inhibitors.

Free heme and the polymerization of sickle cell hemoglobin.

In search of novel control parameters for the polymerization of sickle cell hemoglobin (HbS), the primary pathogenic event of sickle cell anemia, we explore the role of free heme, which may be excessively released in sickle erythrocytes. We show that the concentration of free heme in HbS solutions typically used in the laboratory is 0.02-0.04 mole heme/mole HbS. We show that dialysis of small molecules out of HbS solutions arrests HbS polymerization. The addition of 100-260 µM of free heme to dialyzed HbS solutions leads to rates of nucleation and polymer fiber growth faster by two orders of magnitude than before dialysis. Toward an understanding of the mechanism of nucleation enhancement by heme, we show that free heme at a concentration of 66 µM increases by two orders of magnitude the volume of the metastable clusters of dense HbS liquid, the locations where HbS polymer nuclei form. These results suggest that spikes of the free heme concentration in the erythrocytes of sickle cell anemia patients may be a significant factor in the complexity of the clinical manifestations of sickle cell anemia. The prevention of free heme accumulation in the erythrocyte cytosol may be a novel avenue to sickle cell therapy.

Effect of Erufosine on Membrane Lipid Order in Breast Cancer Cell Models.

Alkylphospholipids are a novel class of antineoplastic drugs showing remarkable therapeutic potential. Among them, erufosine (EPC3) is a promising drug for the treatment of several types of tumors. While EPC3 is supposed to exert its function by interacting with lipid membranes, the exact molecular mechanisms involved are not known yet. In this work, we applied a combination of several fluorescence microscopy and analytical chemistry approaches (i.e., scanning fluorescence correlation spectroscopy, line-scan fluorescence correlation spectroscopy, generalized polarization imaging, as well as thin layer and gas chromatography) to quantify the effect of EPC3 in biophysical models of the plasma membrane, as well as in cancer cell lines. Our results indicate that EPC3 affects lipid-lipid interactions in cellular membranes by decreasing lipid packing and increasing membrane disorder and fluidity. As a consequence of these alterations in the lateral organization of lipid bilayers, the diffusive dynamics of membrane proteins are also significantly increased. Taken together, these findings suggest that the mechanism of action of EPC3 could be linked to its effects on fundamental biophysical properties of lipid membranes, as well as on lipid metabolism in cancer cells.

Study of potential biomedical application of sol-gel derived Zn-doped SiO2-hydroxypropyl cellulose nanohybrids.

A series of Zn-doped hybrid materials based on silica from tetraethoxysilane (TEOS) and hydroxypropyl cellulose (HPC) were prepared by a sol-gel route. The structure, morphology and thermal behavior of synthesized hybrids were characterized by infrared (IR) spectroscopy, ultraviolet-visible spectroscopy (UV-Vis), transmission electron microscopy (TEM) and differential thermal analysis with thermogravimetric analysis (DTA/TG). The obtained materials were investigated for a potential biomedical application. The antibacterial properties of hybrids were investigated by measuring the inhibition zones formed around the materials containing different zinc content in presence of reference strains of Gram-positive and Gram-negative bacteria. The biocompatibility tests showed no cytotoxicity and genotoxicity, as well as no changes in actin cytoskeleton organization for hybrids with Zn content below 5 wt%.

Dimethylsphingosine and miltefosine induce apoptosis in lung adenocarcinoma A549 cells in a synergistic manner.

Lung cancer is one of the most common and lethal types of oncological diseases. Despite the advanced therapeutic approaches, the prognosis for lung cancer still remains poor. Apparently, there is an imperative need for more efficient therapeutic strategies. In this work we report that concurrent treatment of human adenocarcinoma A549 cells with specific concentrations of two antitumor agents, the sphingosine kinase 1 inhibitor N, N dimethylsphingosine (DMS) and the alkylphosphocholine miltefosine, induced synergistic cytotoxic effect, which was confirmed by calculation of the combination index. The simultaneous action of these agents, induced significant decrease of A549 cell number, as well as pronounced morphological alterations. Combined drugs caused substantial apoptotic events, and significant reduction of the pro-survival marker sphingosine- 1-phosphate (S1P), when compared to the individual treatments with each of the anticancer drugs alone. Miltefosine is known to affect the synthesis of choline-containing phospholipids, including sphingomyelin, but we report for the first time that it also reduces S1P. Here we suggest a putative mechanism underlying the effect of miltefosine on sphingosine kinase 1, involving miltefosine-induced inhibition of protein kinase C. In conclusion, our findings provide a possibility for treatment of lung cancer cells with lower concentrations of the two antitumor drugs, DMS and miltefosine, which is favorable, regarding their potential cytotoxicity to normal cells.

New cycloartane triterpenes from bioactive extract of propolis from Pitcairn Island.

Dichloromethane extract of propolis (DCME) originating from Pitcairn Island demonstrated potent cytotoxicity against triple-negative MDA-MB-231 human breast carcinoma cells. The results from MTT assay showed that DCME inhibits the growth of the cancer cells in a dose- and time-dependent manner and upon the cell growth inhibition propolis extract provoked apoptotic changes in the cell nuclei. A detailed chemical investigation of DCME led to the isolation of four new cycloartane triterpenes (1-4), along with 17 known compounds (5-21). The structures of the new compounds were elucidated by means of extensive analysis of their spectroscopic data and comparison with those reported for their analogues. In vitro antimicrobial activity of new compounds (1-4) along with the DCME against four human pathogens was evaluated. All tested constituents except compound 2 were highly active against Escherichia coli with MIC 64 µg/ml. Compound 1 exhibited high antifungal activity against Candida albicans with potency close to that of the positive control (amphotericin B). The DCME showed very good antimicrobial activity against Staphylococcus aureus, Escherichia coli and Candida albicans. This is the first study on propolis from Pitcairn Island.

Phyllostictine A: total synthesis, structural verification and determination of substructure responsible for plant growth inhibition.

The first total synthesis of phyllostictine A (PA) is reported, which confirms the structure of this fungal metabolite and its (6S,7R,8S)-stereochemistry. Both synthetic PA and an analogue containing the 5-methylene-1,5-dihydro-2H-pyrrol-2-one nucleus exhibit µM inhibitory activity in root growth assays against Arabidopsis thaliana, indicating that this heterocyclic subunit is key to the herbicidal activity of the natural product.