RESUMEN
Cyclotides are plant-derived disulfide-rich cyclic peptides that have a natural function in plant defense and potential for use as agricultural pesticides. Because of their highly constrained topology, they are highly resistant to thermal, chemical, or enzymatic degradation. However, the stability of cyclotides at alkaline pH for incubation times of longer than a few days is poorly studied but important since these conditions could be encountered in the environment, during storage or field application as insecticides. In this study, kalata B1 (kB1), the prototypical cyclotide, was engineered to improve its long-term stability and retain its insecticidal activity via point mutations. We found that substituting either Asn29 or Gly1 to lysine or leucine increased the stability of kB1 by twofold when incubated in an alkaline buffer (pH = 9.0) for 7 days, while retaining its insecticidal activity. In addition, when Gly1 was replaced with lysine or leucine, the mutants could be cyclized using an asparaginyl endopeptidase, in vitro with a yield of â¼90% within 5 min. These results demonstrate the potential to manufacture kB1 mutants with increased stability and insecticidal activity recombinantly or in planta. Overall, the discovery of mutants of kB1 that have enhanced stability could be useful in leading to longer term activity in the field as bioinsecticides.
Asunto(s)
Ciclotidas , Insecticidas , Oldenlandia , Ciclotidas/genética , Ciclotidas/farmacología , Ciclotidas/química , Insecticidas/química , Insecticidas/farmacología , Leucina , Lisina/genética , Mutagénesis , Proteínas de Plantas/metabolismo , Oldenlandia/química , Estabilidad Proteica , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacosRESUMEN
Cyclotides are a unique family of stable and cyclic mini-proteins found in plants that have nematicidal and anthelmintic activities. They are distributed across the Rubiaceae, Violaceae, Fabaceae, Cucurbitaceae, and Solanaceae plant families, where they are posited to act as protective agents against pests. In this study, we tested the nematicidal properties of extracts from four major cyclotide-producing plants, Oldenlandia affinis, Clitoria ternatea, Viola odorata, and Hybanthus enneaspermus, against the free-living model nematode Caenorhabditis elegans. We evaluated the nematicidal activity of the cyclotides kalata B1, cycloviolacin O2, and hyen D present in these extracts and found them to be active against the larvae of C. elegans. Both the plant extracts and isolated cyclotides exerted dose-dependent toxicity on the first-stage larvae of C. elegans. Isolated cyclotides caused death or damage upon interacting with the worms' mouth, pharynx, and midgut or membrane. Cycloviolacin O2 and hyen D produced bubble-like structures around the C. elegans membrane, termed blebs, implicating membrane disruption causing toxicity and death. All tested cyclotides lost their toxicity when the hydrophobic patches present on them were disrupted via a single-point mutation. The present results provide a facile assay design to measure and explore the nematicidal activities of plant extracts and purified cyclotides on C. elegans.
Asunto(s)
Ciclotidas , Fabaceae , Nematodos , Violaceae , Animales , Antinematodos/farmacología , Caenorhabditis elegans , Ciclotidas/farmacología , Ciclotidas/química , Fabaceae/química , Extractos Vegetales/química , Proteínas de Plantas/químicaRESUMEN
Cyclotides have a wide range of bioactivities relevant for agricultural and pharmaceutical applications. This large family of naturally occurring macrocyclic peptides is divided into three subfamilies, with the bracelet subfamily being the largest and comprising the most potent cyclotides reported to date. However, attempts to harness the natural bioactivities of bracelet cyclotides and engineer-optimized analogs have been hindered by a lack of understanding of the structural and functional role of their constituent residues, which has been challenging because bracelet cyclotides are difficult to produce synthetically. We recently established a facile strategy to make the I11L mutant of cyclotide hyen D that is as active as the parent peptide, enabling the subsequent production of a series of variants. In the current study, we report an alanine mutagenesis structure-activity study of [I11L] hyen D to probe the role of individual residues on peptide folding using analytical chromatography, on molecular function using surface plasmon resonance, and on therapeutic potential using cytotoxicity assays. We found that Glu-6 and Thr-15 are critical for maintaining the structure of bracelet cyclotides and that hydrophobic residues in loops 2 and 3 are essential for membrane binding and cytotoxic activity, findings that are distinct from the structural and functional characteristics determined for other cyclotide subfamilies. In conclusion, this is the first report of a mutagenesis scan conducted on a bracelet cyclotide, offering insights into their function and supporting future efforts to engineer bracelet cyclotides for biotechnological applications.
Asunto(s)
Ciclotidas , Ciclotidas/química , Ciclotidas/genética , Ciclotidas/toxicidad , Interacciones Hidrofóbicas e Hidrofílicas , Mutagénesis , Unión Proteica/genéticaRESUMEN
Cyclotides have attracted great interest as drug design scaffolds because of their unique cyclic cystine knotted topology. They are classified into three subfamilies, among which the bracelet subfamily represents the majority and comprises the most bioactive cyclotides, but are the most poorly utilized in drug design applications. A long-standing challenge has been the very low in vitro folding yields of bracelets, hampering efforts to characterize their structures and activities. Herein, we report substantial increases in bracelet folding yields enabled by a single point mutation of residue Ile-11 to Leu or Gly. We applied this discovery to synthesize mirror image enantiomers and used quasi-racemic crystallography to elucidate the first crystal structures of bracelet cyclotides. This study provides a facile strategy to produce bracelet cyclotides, leading to a general method to easily access their atomic resolution structures and providing a basis for development of biotechnological applications.
Asunto(s)
Ciclotidas , Secuencia de Aminoácidos , Cristalografía , Cistina , Pliegue de ProteínaRESUMEN
Hybanthus enneaspermus is an Indian folk medicinal herb that has been widely used as a libido enhancer. This plant belongs to the Violaceae plant family, which ubiquitously contains disulfide-rich cyclic peptides named cyclotides. Cyclotides are an expanding plant-derived peptide family with numerous interesting bioactivities, and their unusual stability against proteolysis has attracted much attention in drug design applications. Recently, H. enneaspermus has been reported to be a rich source of cyclotides, and hence, it was of interest to investigate whether cyclotides contribute to its aphrodisiac activity. In this study, we evaluated the in vivo aphrodisiac activity of the herbal powder, extract, and the most abundant cyclotide, hyen D, extracted from H. enneaspermus on rats in a single dose regimen. After dosing, the sexual behaviors of male rats were observed, recorded, analyzed, and compared with those of the vehicle group. The results show that the extract and hyen D significantly decreased the intromission latency of sexually naïve male rats and the extract improved a range of other measured sexual parameters. The results suggest that the extract could enhance libido as well as facilitate erectile function in male rats and that the cyclotide hyen D could contribute to the libido-enhancing activity of this ethnomedicinal herb.
Asunto(s)
Afrodisíacos/farmacología , Extractos Vegetales/farmacología , Violaceae/química , Animales , Femenino , Masculino , Ratas , Conducta Sexual AnimalRESUMEN
Cyclotides are plant-derived peptides characterized by an â¼30-amino acid-long cyclic backbone and a cystine knot motif. Cyclotides have diverse bioactivities, and their cytotoxicity has attracted significant attention for its potential anticancer applications. Hybanthus enneaspermus (Linn) F. Muell is a medicinal herb widely used in India as a libido enhancer, and a previous study has reported that it may contain cyclotides. In the current study, we isolated 11 novel cyclotides and 1 known cyclotide (cycloviolacin O2) from H. enneaspermus and used tandem MS to determine their amino acid sequences. We found that among these cyclotides, hyen C comprises a unique sequence in loops 1, 2, 3, 4, and 6 compared with known cyclotides. The most abundant cyclotide in this plant, hyen D, had anticancer activity comparable to that of cycloviolacin O2, one of the most cytotoxic known cyclotides. We also provide mechanistic insights into how these novel cyclotides interact with and permeabilize cell membranes. Results from surface plasmon resonance experiments revealed that hyen D, E, L, and M and cycloviolacin O2 preferentially interact with model lipid membranes that contain phospholipids with phosphatidyl-ethanolamine headgroups. The results of a lactate dehydrogenase assay indicated that exposure to these cyclotides compromises cell membrane integrity. Using live-cell imaging, we show that hyen D induces rapid membrane blebbing and cell necrosis. Cyclotide-membrane interactions correlated with the observed cytotoxicity, suggesting that membrane permeabilization and disintegration underpin cyclotide cytotoxicity. These findings broaden our knowledge on the indigenous Indian herb H. enneaspermus and have uncovered cyclotides with potential anticancer activity.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Ciclotidas/farmacología , Descubrimiento de Drogas , Plantas Medicinales/química , Violaceae/química , Secuencia de Aminoácidos , Antineoplásicos Fitogénicos/química , Antineoplásicos Fitogénicos/aislamiento & purificación , Línea Celular Tumoral , Ciclotidas/química , Ciclotidas/aislamiento & purificación , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Proteínas de Plantas/química , Proteínas de Plantas/aislamiento & purificación , Proteínas de Plantas/farmacología , Resonancia por Plasmón de Superficie , Espectrometría de Masas en TándemRESUMEN
Cyclotides are a plant-derived family of peptides that comprise approximately 30â¯amino acid residues, a cyclic backbone and a cystine knot. Due to their unique structure, cyclotides are exceptionally stable to heat or proteolytic degradation and are tolerant to amino acid substitutions in their backbone loops between conserved cysteine residues. Their toxicity to insect pests and their make-up of natural amino acids has led to their applications in eco-friendly crop protection. Furthermore, their stability and cell penetrating properties make cyclotides ideal scaffolds for bioactive epitope grafting. This article gives a brief overview of cyclotide discovery, characterization, distribution, synthesis and mode of action mechanisms. We focus on their toxicities to insect pests and their medical and agricultural applications.
Asunto(s)
Ciclotidas/química , Magnoliopsida/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Ciclotidas/farmacología , Ciclotidas/toxicidad , Insectos/efectos de los fármacos , Toxinas Biológicas/química , Toxinas Biológicas/toxicidadRESUMEN
Isosteroidal alkaloids are a category of promising bioactive compounds which mostly exist in plants of genus Veratrum and Fritillaria. The pharmacological activities of isosteroidal alkaloids include antihypertensive, antitussive, anti-inflammatory, antithrombosis, among others. Recently, some studies show that this kind of alkaloids exhibited significant antitumor activity. To the best of our knowledge, there is no review focusing on their antitumor activity and mechanism of their antitumor activity. To fill the gap, in this review, we summarized antitumor effects of the isosteroidal alkaloids from genus Veratrum and Fritillaria on different tumors and the mechanisms of their antitumor activity. In conclusion, this kind of alkaloids has extensive antitumor activity, and there are several main mechanisms of their antitumor activity, including the Hedgehog signaling pathway, caspase-3 dependent apoptosis, cell cycle, and autophagy.
Asunto(s)
Alcaloides/química , Alcaloides/farmacología , Antineoplásicos Fitogénicos/química , Antineoplásicos Fitogénicos/farmacología , Fritillaria/química , Neoplasias/tratamiento farmacológico , Veratrum/química , Alcaloides/uso terapéutico , Animales , Antineoplásicos Fitogénicos/uso terapéutico , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Proteínas Hedgehog/metabolismo , Humanos , Neoplasias/metabolismo , Neoplasias/patología , Transducción de Señal/efectos de los fármacos , Alcaloides de Veratrum/química , Alcaloides de Veratrum/farmacología , Alcaloides de Veratrum/uso terapéuticoRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Bulb of Fritillaria cirrhosa D.Don (BFC) has been wildly used in China for a long time for folk medicine since its significant therapeutic effects on respiratory diseases, such as cough, expectoration, pneumonia and bronchial inflammation, which are related to respiratory inflammatory response. However, there is a lack of investigation on the in vivo anti-inflammatory properties of BFC. AIM OF THE STUDY: The aim of this study was to evaluate the in vivo anti-inflammatory activity of the purified total alkaloid fraction of BFC (TAF) by using different animal models of inflammation to provide scientific evidence for its traditional use. MATERIALS AND METHODS: The total alkaloid fraction from BFC was prepared by using H-103 resin column. Anti-inflammatory effect of TAF was evaluated by models of acetic acid-induced capillary permeability accentuation, carrageenan-induced rat paw edema, cotton pellet-induced granuloma formation and LPS-induced acute lung injury (ALI). The level of cytokines (TNF, IL-6, IL-4 and IL-10) was measured by ELISA. Histopathological analyses were performed by using hematoxylin and eosin staining. RESULTS: TAF can inhibit acetic acid-induced capillary permeability accentuation, carrageenan-induced paw edema, cotton pellet-induced granuloma formation, suppress inflammatory cells recruitment and cytokine production in the bronchoalveolar lavage fluid from LPS-induced ALI mice, and attenuate pathological changes in the lung tissues of ALI mice. CONCLUSION: This study provides scientific evidence for bulb of F. cirrhosa to treat respiratory inflammation.
Asunto(s)
Lesión Pulmonar Aguda/prevención & control , Alcaloides/farmacología , Antiinflamatorios/farmacología , Fritillaria/química , Extractos Vegetales/farmacología , Animales , Líquido del Lavado Bronquioalveolar , Citocinas/metabolismo , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratas , Ratas Sprague-DawleyRESUMEN
Chronic obstructive pulmonary disease (COPD) is the third leading cause of death in the world. Present therapies for COPD have limited effect on reducing the progression of COPD and suppressing the inflammatory response in the lung. Bulbs of Fritillaria cirrhosa D. Don (BFC) have been used in many Asian countries for a long time to treat pulmonary diseases, such as cough, expectoration, and asthma. Steroidal alkaloids are the major biological active constituents in BFC, whereby imperialine is one of the important steroidal alkaloids. So far, there are no studies reporting the effect of imperialine on COPD. In this study, we investigated the effect of imperialine on pulmonary function and structure and inflammation in a COPD-like rat model which was induced by the combination of exposure to CS and intratracheal administration of LPS. Our data show that imperialine mitigates pulmonary functional and structural impairment and suppressed inflammatory response in a COPD-like rat model by mediating expression of related cytokines in lung tissues of the COPD-like rats, such as IL-1ß, IL-6, IL-8, TNF-α, NF-κB, TGF-ß1, MMP-9, and TIMP-1.
Asunto(s)
Alcaloides/farmacología , Fritillaria/química , Inflamación/tratamiento farmacológico , Pulmón/efectos de los fármacos , Extractos Vegetales/farmacología , Enfermedad Pulmonar Obstructiva Crónica/fisiopatología , Animales , Antiinflamatorios/farmacología , Peso Corporal , Cevanas/metabolismo , Citocinas/metabolismo , Modelos Animales de Enfermedad , Inmunohistoquímica , Pulmón/metabolismo , Masculino , Raíces de Plantas/química , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Ratas , Ratas Wistar , Pruebas de Función Respiratoria , Esteroides/metabolismoRESUMEN
The bulbs of cultivated Fritillaria cirrhosa (BCFC) are used in China both for food and folk medicine due to its powerful biological activities. The aim of this study is to optimize the extraction and enrichment conditions of alkaloids from BCFC. Firstly, the orthogonal experimental design was used to optimize and evaluate four variables (ethanol concentration, solid-liquid ratio, extraction time, and temperature). Thereafter, resin adsorption was as a means to enrich alkaloids. Among 16 tested resins, H-103 resin presented higher adsorption capacity and desorption ratio. The equilibrium experimental data of the adsorption of total alkaloids, imperialine, and peimisine were well-fitted to the pseudo-first-order kinetics model, Langmuir and Freundlich isotherms models. Finally, in order to optimize the parameters for purifying alkaloids, dynamic adsorption and desorption tests were carried out. After one run treatment with H-103 resin, the contents of total alkaloids, imperialine, and peimisine in the product were 21.40-, 18.31-, and 22.88-fold increased with recovery yields of 94.43%, 90.57%, and 96.16%, respectively.