RESUMEN
In the Americas and specially in Brazil, the Loxosceles intermedia, Loxosceles gaucho and Loxosceles laeta are the three most medically relevant brown spider species, and whose bites can lead to the condition known as loxoscelism. Here, we report the development of a tool capable of identifying a common epitope amongst Loxosceles sp. venom's toxins. A murine monoclonal antibody (LmAb12) and its recombinant fragments (scFv12P and diabody12P) have been produced and characterized. This antibody and its recombinant constructs were able to recognize proteins of Loxosceles spider venoms with specificity. The scFv12P variant was also able to detect low concentrations of Loxosceles venom in a competitive ELISA assay, displaying potential as a venom identification tool. The primary antigenic target of LmAb12 is a knottin, a venom neurotoxin, that has a shared identity of 100 % between the L. intermedia and L. gaucho species and high similarity to L. laeta. Furthermore, we observed LmAb12 was able to partially inhibit in vitro hemolysis, a cellular event typically induced by the Loxosceles sp. venoms. Such behavior might be due to LmAb12 cross-reactivity between the antigenic target of LmAb12 and the venom's dermonecrotic toxins, the PLDs, or even the existence of synergism between these two toxins.
Asunto(s)
Venenos de Araña , Arañas , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Antígenos/química , Antivenenos/química , Reacciones Cruzadas , Miniproteínas Nodales de Cistina/química , Fosfolipasa D/química , Venenos de Araña/química , Arañas/química , Epítopos/químicaRESUMEN
The extra domain B splice variant (EDB) of human fibronectin selectively expressed in the tumor vasculature is an attractive target for cancer imaging and therapy. Here, we describe the generation and characterization of EDB-specific optical imaging probes. By screening combinatorial cystine-knot miniprotein libraries with phage display technology we discover exquisitely EDB-specific ligands that share a distinctive motif. Probes with a binding constant in the picomolar range are generated by chemical oligomerization of selected ligands and fluorophore conjugation. We show by fluorescence imaging that the probes stain EDB in tissue sections derived from human U-87 MG glioblastoma xenografts in mice. Moreover, we demonstrate selective accumulation and retention of intravenously administered probes in the tumor tissue of mice with U-87 MG glioblastoma xenografts by in vivo and ex vivo fluorescence imaging. These data warrants further pursuit of the selected cystine-knot miniproteins for in vivo imaging applications.
Asunto(s)
Miniproteínas Nodales de Cistina/metabolismo , Fibronectinas/metabolismo , Glioblastoma/irrigación sanguínea , Proteínas Recombinantes/metabolismo , Secuencias de Aminoácidos , Animales , Sitios de Unión , Línea Celular Tumoral , Miniproteínas Nodales de Cistina/química , Miniproteínas Nodales de Cistina/genética , Miniproteínas Nodales de Cistina/uso terapéutico , Fibronectinas/genética , Colorantes Fluorescentes/química , Colorantes Fluorescentes/uso terapéutico , Glioblastoma/diagnóstico por imagen , Glioblastoma/metabolismo , Humanos , Ratones , Ratones Desnudos , Imagen Óptica , Biblioteca de Péptidos , Ingeniería de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Resonancia por Plasmón de Superficie , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Nerve growth factor (NGF) is an endogenously produced polypeptide that promotes the differentiation, survival, and repair of neurons in the central and peripheral nervous systems. While trophic proteins hold promise for the treatment of neuronal injury and disease, use of NGF is limited by its large molecular weight, lack of permeability through the blood-brain barrier, and peripheral side effects. Previously, we found that an extract of the Momordica cochinchinensis seed stimulated PC-12 neurite outgrowth. Bioactivity-guided fractioning of the seed extract suggested that the NGF mimetic agent was one of few defined proteins from this plant: one group being the defense Knottins and the other group of the lowest mass is the potent trypsin inhibitor MCoTI-II. Here, the NGF mimetic potential of this latter protein was investigated using two concurrent but different approaches. A biological study used recombinant purified MCoTI-II, which when tested in rat PC-12 cells grown on collagen, failed to initiate outgrowth relative to the positive control 7S NGF. In a separate computational study, the possibility was investigated such that MCoTI-II could exert an effect through binding to the serine protease γ-NGF subunit of the 7S NGF complex, analogous to its binding to its native receptor trypsin. Molecular dynamics simulations showed that MCoTI-II can bind stably to γ-NGF for >350 ns. Modeling indicated that this interaction could sterically inhibit 7S NGF complex formation, potentially altering the equilibrium between inactive complexed and free active NFG protein. In conclusion, the biological study now excludes the MCoTI-II protein as the NGF mimetic factor in the Momordica extract, an important and required step to identify the active component in this seed. On the other hand, the theoretical study has revealed a novel observation that may be of use in the development of strategies to affect NGF activity.
Asunto(s)
Ciclotidas/metabolismo , Miniproteínas Nodales de Cistina/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Proyección Neuronal , Extractos Vegetales/metabolismo , Animales , Biomimética , Simulación por Computador , Ciclotidas/química , Miniproteínas Nodales de Cistina/química , Simulación de Dinámica Molecular , Momordica/química , Factores de Crecimiento Nervioso/química , Células PC12 , Unión Proteica , Conformación Proteica , Ratas , Serina Endopeptidasas/metabolismoRESUMEN
Gating modifier toxins from spider venom are disulfide-rich peptides that typically comprise a stabilizing inhibitor cystine knot (ICK). These knottin peptides are being pursued as therapeutic leads for a range of conditions linked to transmembrane proteins. Recently, double-knottin peptides discovered in spider venom and produced by recombinant expression have provided insights into the pharmacology of transmembrane channels. Here, we use chemoenzymatic ligation to produce double-knottins to probe the effect of bivalent modulation on the voltage-gated sodium channel subtype 1.7 (NaV1.7), which is implicated in pain signaling. Monovalent knottins were oxidatively folded and then biochemically conjugated using sortase A, to form double-knottins. The structural integrity of the peptides was confirmed using NMR, and fluorescence-based activity assays provided evidence suggesting that coincubated monovalent and bivalent knottins can cooperatively modulate NaV1.7. We anticipate that double-knottins will provide novel tools for enhancing our understanding of, and design strategies for, therapeutically relevant voltage-gated ion channels.
Asunto(s)
Aminoaciltransferasas/química , Proteínas Bacterianas/química , Cisteína Endopeptidasas/química , Miniproteínas Nodales de Cistina/química , Venenos de Araña/química , Canales de Sodio Activados por Voltaje/química , Secuencia de Aminoácidos , Membrana Dobles de Lípidos/química , Sondas Moleculares , Resonancia Magnética Nuclear Biomolecular , Resonancia por Plasmón de SuperficieRESUMEN
A big hurdle for the use of protein-based drugs is that they are easily degraded by proteases in the human body. In an attempt to solve this problem, we show the possibility to functionalize TM encapsulin nanoparticles with an mEETI-II knottin miniprotein from the cysteine-stabilized knot class. The resulting particles did not show aggregation and retained part of their protease inhibitive function. This imposes a protection toward protease, in this case, trypsin, degradation of the protein cage. The used chemistry is easy to apply and thus suitable to protect other protein systems from degradation. In addition, this proof of principle opens up the use of other knottins or cysteine-stabilized knots, which can be attached to protein cages to create a heterofunctionalized protein nanocage. This allows specific targeting and tumor suppression among other types of functionalization. Overall, this is a promising strategy to protect a protein of interest which brings oral administration of protein-based drugs one step closer.
Asunto(s)
Miniproteínas Nodales de Cistina/química , Portadores de Fármacos/química , Inhibidores de Tripsina/química , Tripsina/metabolismo , Administración Oral , Proteínas Bacterianas/administración & dosificación , Proteínas Bacterianas/metabolismo , Miniproteínas Nodales de Cistina/farmacología , Portadores de Fármacos/farmacología , Nanopartículas/química , Proteolisis , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/metabolismo , Thermotoga maritima , Inhibidores de Tripsina/farmacologíaRESUMEN
Cystine-knot miniproteins (CKMPs) are an intriguing group of cysteine-rich molecules that combine the characteristics of proteins and peptides. Typically, CKMPs are fewer than 50 residues in length and share a characteristic knotted scaffold characterized by the presence of three intramolecular disulfide bonds that form the singular knotted structure. The knot scaffold confers on these proteins remarkable chemical, thermal, and proteolytic stability. Recently, CKMPs have emerged as a novel class of natural molecules with interesting pharmacological properties. In the present work, a novel cystine-knot metallocarboxypeptidase inhibitor (chuPCI) was isolated from tubers of Solanum tuberosum, subsp. andigenum cv. Churqueña. Our results demonstrated that chuPCI is a member of the A/B-type family of metallocarboxypeptidases inhibitors. chuPCI was expressed and characterized by a combination of biochemical and mass spectrometric techniques. Direct comparison of the MALDI-TOF mass spectra for the native and recombinant molecules allowed us to confirm the presence of four different forms of chuPCI in the tubers. The majority of such forms have a molecular weight of 4309 Da and contain a cyclized Gln in the N-terminus. The other three forms are derived from N-terminal and/or C-terminal proteolytic cleavages. Taken together, our results contribute to increase the current repertoire of natural CKMPs.
Asunto(s)
Miniproteínas Nodales de Cistina/química , Proteínas de Plantas/química , Proteómica , Proteínas Recombinantes , Solanum tuberosum/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Secuencia de Aminoácidos , Animales , Carboxipeptidasas/antagonistas & inhibidores , Bovinos , Clonación Molecular , Miniproteínas Nodales de Cistina/análisis , Miniproteínas Nodales de Cistina/genética , Miniproteínas Nodales de Cistina/aislamiento & purificación , Activación Enzimática/efectos de los fármacos , Cinética , Proteínas de Plantas/análisis , Proteínas de Plantas/genética , Proteínas de Plantas/aislamiento & purificación , Inhibidores de Proteasas/análisis , Inhibidores de Proteasas/química , Inhibidores de Proteasas/aislamiento & purificación , Inhibidores de Proteasas/farmacología , Proteómica/métodos , Análisis de Secuencia de ADN , Solanum tuberosum/genética , Solanum tuberosum/metabolismo , PorcinosRESUMEN
Knottins, or inhibitor cystine knots (ICKs), are ultra-stable miniproteins with multiple applications in drug design and medical imaging. These widespread and functionally diverse proteins are characterized by the presence of three interwoven disulfide bridges in their structure, which form a unique pseudoknot. Since 2004, the KNOTTIN database (www.dsimb.inserm.fr/KNOTTIN/) has been gathering standardized information about knottin sequences, structures, functions and evolution. The website also provides access to bibliographic data and to computational tools that have been specifically developed for ICKs. Here, we present a major upgrade of our database, both in terms of data content and user interface. In addition to the new features, this article describes how KNOTTIN has seen its size multiplied over the past ten years (since its last publication), notably with the recent inclusion of predicted ICKs structures. Finally, we report how our web resource has proved usefulness for the researchers working on ICKs, and how the new version of the KNOTTIN website will continue to serve this active community.
Asunto(s)
Miniproteínas Nodales de Cistina/química , Bases de Datos de Proteínas/historia , Modelos Moleculares , Interfaz Usuario-Computador , Secuencia de Aminoácidos , Gráficos por Computador , Miniproteínas Nodales de Cistina/clasificación , Miniproteínas Nodales de Cistina/genética , Miniproteínas Nodales de Cistina/metabolismo , Disulfuros , Expresión Génica , Historia del Siglo XXI , Humanos , Internet , Ligandos , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Alineación de Secuencia , Análisis de Secuencia de ProteínaRESUMEN
We describe a highly sensitive competition ELISA to measure integrin-binding of RGD-peptides in high-throughput without using cells, ECM-proteins, or antibodies. The assay measures (nonlabeled) RGD-peptides' ability to inhibit binding of a biotinylated "knottin"-RGD peptide to surface-immobilized integrins and, thus, enables quantification of the binding strength of high-, medium-, and low-affinity RGD-binders. We introduced the biotinylated knottin-RGD peptide instead of biotinylated cyclo[RGDfK] (as reported by Piras et al.), as integrin-binding was much stronger and clearly detectable for all three integrins. In order to maximize sensitivity and cost-efficiency, we first optimized several parameters, such as integrin-immobilization levels, knottin-RGD concentration, buffer compositions, type of detection tag (biotin, His- or cMyc-tag), and spacer length. We thereby identified two key factors, that is, (i) the critical spacer length (longer than Gly) and (ii) the presence of Ca2+ and Mg2+ in all incubation and washing buffers. Binding of knottin-RGD peptide was strongest for αvß3 but also detectable for both αvß5 and α5ß1, while binding of biotinylated cyclo[RGDfK] was very weak and only detectable for αvß3. For assay validation, we finally determined IC50 values for three unlabeled peptides, that is: (i) linear GRGDS, (ii) cyclo[RGDfK], and (iii) the knottin-RGD itself for binding to three different integrin receptors (αvß3, αvß5, α5ß1). Major benefits of the novel assay are (i) the extremely low consumption of integrin (50 ng/peptide), (ii) the fact that neither antibodies/ECM-proteins nor integrin-expressing cells are required for detection, and (iii) its suitability for high-throughput screening of (RGD-)peptide libraries.
Asunto(s)
Miniproteínas Nodales de Cistina/metabolismo , Ensayos Analíticos de Alto Rendimiento , Oligopéptidos , Péptidos/metabolismo , Biotinilación , Miniproteínas Nodales de Cistina/química , Integrina alfa5beta1/antagonistas & inhibidores , Integrina alfa5beta1/metabolismo , Integrina alfaVbeta3/antagonistas & inhibidores , Integrina alfaVbeta3/metabolismo , Biblioteca de Péptidos , Péptidos/química , Unión Proteica , Receptores de Vitronectina/antagonistas & inhibidores , Receptores de Vitronectina/metabolismoRESUMEN
Loxosceles intermedia venom comprises a complex mixture of proteins, glycoproteins and low molecular mass peptides that act synergistically to immobilize envenomed prey. Analysis of a venom-gland transcriptome from L. intermedia revealed that knottins, also known as inhibitor cystine knot peptides, are the most abundant class of toxins expressed in this species. Knottin peptides contain a particular arrangement of intramolecular disulphide bonds, and these peptides typically act upon ion channels or receptors in the insect nervous system, triggering paralysis or other lethal effects. Herein, we focused on a knottin peptide with 53 amino acid residues from L. intermedia venom. The recombinant peptide, named U2 -sicaritoxin-Li1b (Li1b), was obtained by expression in the periplasm of Escherichia coli. The recombinant peptide induced irreversible flaccid paralysis in sheep blowflies. We screened for knottin-encoding sequences in total RNA extracts from two other Loxosceles species, Loxosceles gaucho and Loxosceles laeta, which revealed that knottin peptides constitute a conserved family of toxins in the Loxosceles genus. The insecticidal activity of U2 -SCTX-Li1b, together with the large number of knottin peptides encoded in Loxosceles venom glands, suggests that studies of these venoms might facilitate future biotechnological applications of these toxins.
Asunto(s)
Araña Reclusa Parda/genética , Miniproteínas Nodales de Cistina/química , Insecticidas/análisis , Hidrolasas Diéster Fosfóricas/química , Venenos de Araña/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Araña Reclusa Parda/metabolismo , Secuencia Conservada , Miniproteínas Nodales de Cistina/biosíntesis , Miniproteínas Nodales de Cistina/genética , Miniproteínas Nodales de Cistina/aislamiento & purificación , Dípteros , Electroforesis en Gel de Poliacrilamida , Escherichia coli , Datos de Secuencia Molecular , Proteoma , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Pruebas de Toxicidad , TranscriptomaRESUMEN
Inhibitor cystine-knots, also known as knottins, are a structural family of ultra-stable peptides with diverse functions. Knottins and related backbone-cyclized peptides called cyclotides contain three disulfide bonds connected in a particular arrangement that endows these peptides with high thermal, proteolytic, and chemical stability. Knottins have gained interest as candidates for non-invasive molecular imaging and for drug development as they can possess the pharmacological properties of small molecules and the target affinity and selectively of protein biologics. Naturally occurring knottins are clinically approved for treating chronic pain and GI disorders. Combinatorial methods are being used to engineer knottins that can bind to other clinically relevant targets in cancer, and inflammatory and cardiac disease. This review details recent examples of engineered knottin peptides; their use as molecular imaging agents, therapeutics, and drug delivery vehicles; modifications that can be introduced to improve peptide folding and bioactivity; and future perspectives and challenges in the field.
Asunto(s)
Miniproteínas Nodales de Cistina/química , Diagnóstico , Portadores de Fármacos , Péptidos/química , Terapéutica , Animales , Humanos , Ingeniería de ProteínasRESUMEN
Plant seed knottins, mainly from the Cucurbitacea family, and sunflower seed trypsin inhibitor (SFTI 1) are the most low-molecular canonical peptide inhibitors of serine proteases. High efficiency of inhibition of various serine proteases, structure rigidity together with the possibility of limited variations of amino acid sequences, high chemical stability, lack of toxic properties, opportunity of production by either chemical synthesis or use of heterologous expression systems make these inhibitors attractive templates for design of new compounds for regulation of therapeutically significant serine protease activities. Hence the design of such compounds represents a prospective research field. The review considers structural characteristics of these inhibitors, their properties, methods of preparation and design of new analogs. Examples of successful employment of natural serine protease inhibitors belonging to knottin family and SFTI 1 as templates for the design of highly specific inhibitors of certain proteases are given.
Asunto(s)
Miniproteínas Nodales de Cistina/química , Péptidos Cíclicos/farmacología , Proteínas de Plantas/química , Inhibidores de Serina Proteinasa/farmacología , Animales , Miniproteínas Nodales de Cistina/metabolismo , Descubrimiento de Drogas , Humanos , Péptidos Cíclicos/síntesis química , Péptidos Cíclicos/química , Proteínas de Plantas/metabolismo , Unión Proteica , Inhibidores de Serina Proteinasa/síntesis química , Inhibidores de Serina Proteinasa/químicaRESUMEN
Inhibitor cystine knots (ICKs) are a family of structural peptides with a large number of cysteine residues that form intramolecular disulfide bonds, resulting in a knot. These peptides are involved in a variety of biological functions including predation and defense, and are found in various species, such as spiders, scorpions, sea anemones, and plants. The Loxosceles intermedia venom gland transcriptome identified five groups of ICK peptides that represent more than 50 % of toxin-coding transcripts. Here, we describe the molecular cloning of U2-Sicaritoxin-Lit2 (U2-SCRTX-Lit2), bioinformatic characterization, structure prediction, and molecular dynamic analysis. The sequence of U2-SCRTX-Lit2 obtained from the transcriptome is similar to that of µ-Hexatoxin-Mg2, a peptide that inhibits the insect Nav channel. Bioinformatic analysis of sequences classified as ICK family members also showed a conservation of cysteine residues among ICKs from different spiders, with the three dimensional molecular model of U2-SCRTX-Lit2 similar in structure to the hexatoxin from µ-hexatoxin-Mg2a. Molecular docking experiments showed the interaction of U2-SCRTX-Lit2 to its predictable target-the Spodoptera litura voltage-gated sodium channel (SlNaVSC). After 200 ns of molecular dynamic simulation, the final structure of the complex showed stability in agreement with the experimental data. The above analysis corroborates the existence of a peptide toxin with insecticidal activity from a novel ICK family in L. intermedia venom and demonstrates that this peptide targets Nav channels.
Asunto(s)
Miniproteínas Nodales de Cistina/química , Modelos Moleculares , Venenos de Araña/química , Arañas/química , Secuencia de Aminoácidos , Animales , Clonación Molecular , Simulación del Acoplamiento Molecular , Estructura Terciaria de ProteínaRESUMEN
Antibody-drug conjugates (ADCs) offer increased efficacy and reduced toxicity compared to systemic chemotherapy. Less attention has been paid to peptide-drug delivery, which has the potential for increased tumor penetration and facile synthesis. We report a knottin peptide-drug conjugate (KDC) and demonstrate that it can selectively deliver gemcitabine to malignant cells expressing tumor-associated integrins. This KDC binds to tumor cells with low-nanomolar affinity, is internalized by an integrin-mediated process, releases its payload intracellularly, and is a highly potent inhibitor of brain, breast, ovarian, and pancreatic cancer cell lines. Notably, these features enable this KDC to bypass a gemcitabine-resistance mechanism found in pancreatic cancer cells. This work expands the therapeutic relevance of knottin peptides to include targeted drug delivery, and further motivates efforts to expand the drug-conjugate toolkit to include non-antibody protein scaffolds.
Asunto(s)
Antineoplásicos/farmacología , Miniproteínas Nodales de Cistina/metabolismo , Desoxicitidina/análogos & derivados , Integrinas/antagonistas & inhibidores , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Miniproteínas Nodales de Cistina/química , Desoxicitidina/química , Desoxicitidina/farmacología , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Conformación Molecular , Relación Estructura-Actividad , GemcitabinaRESUMEN
Two new cystine knot peptides, asteropsins F (ASPF) and G (ASPG), were isolated from the marine sponge Asteropus sp. ASPF and ASPG are composed of 33 and 32 amino acids, respectively, and contain six cysteines which are involved in three disulfide bonds. They shared the characteristic features of the asteropsin family, such as, N-terminal pyroglutamate modification, incorporation of cis prolines, and the unique anionic profile, which distinguish them from other knottin families. Tertiary structures of the peptides were determined by high resolution NMR. ASPF and ASPG were found to be remarkably resistant not only to digestive enzymes (chymotrypsin, pepsin, elastase, and trypsin) but also to thermal degradation. In addition, these peptides were pharmacologically inert; non-hemolytic to human and fish red blood cells, non-stimulatory to murine macrophage cells, and nontoxic in vitro or in vivo. These observations support their stability and biocompatibility as suitable carrier scaffolds for the design of oral peptide drug.
Asunto(s)
Materiales Biocompatibles/química , Miniproteínas Nodales de Cistina/química , Péptidos/química , Poríferos/química , Secuencia de Aminoácidos , Animales , Materiales Biocompatibles/aislamiento & purificación , Materiales Biocompatibles/farmacología , Línea Celular , Miniproteínas Nodales de Cistina/farmacología , Eritrocitos/efectos de los fármacos , Peces , Humanos , Macrófagos/efectos de los fármacos , Espectroscopía de Resonancia Magnética , Ratones , Modelos Moleculares , Péptidos/genética , Estabilidad Proteica , Estructura Terciaria de Proteína , Alineación de Secuencia , TemperaturaRESUMEN
Antibody-drug conjugates (ADC) have generated significant interest as targeted therapeutics for cancer treatment, demonstrating improved clinical efficacy and safety compared with systemic chemotherapy. To extend this concept to other tumor-targeting proteins, we conjugated the tubulin inhibitor monomethyl-auristatin-F (MMAF) to 2.5F-Fc, a fusion protein composed of a human Fc domain and a cystine knot (knottin) miniprotein engineered to bind with high affinity to tumor-associated integrin receptors. The broad expression of integrins (including αvß3, αvß5, and α5ß1) on tumor cells and their vasculature makes 2.5F-Fc an attractive tumor-targeting protein for drug delivery. We show that 2.5F-Fc can be expressed by cell-free protein synthesis, during which a non-natural amino acid was introduced into the Fc domain and subsequently used for site-specific conjugation of MMAF through a noncleavable linker. The resulting knottin-Fc-drug conjugate (KFDC), termed 2.5F-Fc-MMAF, had approximately 2 drugs attached per KFDC. 2.5F-Fc-MMAF inhibited proliferation in human glioblastoma (U87MG), ovarian (A2780), and breast (MB-468) cancer cells to a greater extent than 2.5F-Fc or MMAF alone or added in combination. As a single agent, 2.5F-Fc-MMAF was effective at inducing regression and prolonged survival in U87MG tumor xenograft models when administered at 10 mg/kg two times per week. In comparison, tumors treated with 2.5F-Fc or MMAF were nonresponsive, and treatment with a nontargeted control, CTRL-Fc-MMAF, showed a modest but not significant therapeutic effect. These studies provide proof-of-concept for further development of KFDCs as alternatives to ADCs for tumor targeting and drug delivery applications. Mol Cancer Ther; 15(6); 1291-300. ©2016 AACR.
Asunto(s)
Miniproteínas Nodales de Cistina/química , Inmunoconjugados/farmacología , Integrinas/metabolismo , Neoplasias/tratamiento farmacológico , Oligopéptidos/farmacología , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Sistema Libre de Células , Sistemas de Liberación de Medicamentos , Humanos , Inmunoconjugados/química , Fragmentos Fc de Inmunoglobulinas/química , Integrinas/química , Ratones , Oligopéptidos/química , Péptidos/química , Unión Proteica , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
MCoTI-I and II are plant defence proteins, potent trypsin inhibitors from the bitter gourd Momordica cochinchinensis. They are members of the Knottin Family, which display exceptional stability due to unique topology comprising three interlocked disulfide bridges. Knottins show promise as scaffolds for new drug development. A crystal structure of trypsin-bound MCoTI-II suggested that loop 1, which engages the trypsin active site, would show decreased dynamics in the bound state, an inference at odds with an NMR analysis of MCoTI-I, which revealed increased dynamics of loop 1 in the presence of trypsin. To investigate this question, we performed unrestrained MD simulations of trypsin-bound and free MCoTI-II. This analysis found that loop 1 of MCoTI-II is not more dynamic in the trypsin-bound state than in the free state. However, it revealed an intermediate conformation, transitional between the free and bound MCoTI-II states. The data suggest that MCoTI-II binding involves a process in which initial interaction with trypsin induces transitions between the free and intermediate conformations, and fluctuations between these states account for the increase in dynamics of loop 1 observed for trypsin-bound MCoTI-I. The MD analysis thus revealed new aspects of the inhibitors' dynamics that may be of utility in drug design.
Asunto(s)
Ciclotidas/química , Miniproteínas Nodales de Cistina/química , Proteínas de Plantas/química , Inhibidores de Tripsina/química , Tripsina/química , Simulación de Dinámica Molecular , Unión Proteica , Conformación ProteicaRESUMEN
Knottins are known as a robust and versatile class of miniprotein scaffolds for the presentation of high-affinity binding peptides; however, to date their application in biomaterials, biological coatings, and surface applications have not been explored. We have developed a strategy to recombinantly synthesize a ß-trypsin inhibitory knottin with supramolecular guest tags that enable it to adhere to self-assembled monolayers of the supramolecular host cucurbit[8]uril (CB[8]). We have described a strategy to easily express knottins in E. coli by conjugating them to a fluorescent protein after which they are cleaved and purified. Knottin constructs that varied in the number and position of the supramolecular tag at either the N- or C-termini or at both ends have been verified for their trypsin inhibitory function and CB[8]-binding properties in solution and on surfaces. All of the knottin constructs showed strong inhibition of trypsin with inhibition constants between 10 and 30 nM. Using microscale thermophoresis, we determined that the supramolecular guest tags on the knottins bind CB[8] with a Kd of â¼6 µM in solution. At the surface, strong divalent binding has been determined with a Kd of 0.75 µM in the case of the knottin with two supramolecular guest tags, whereas only weak monovalent binding occurred when only one guest tag was present. We also show successful supramolecular surface immobilization of the knottin using CB[8] and prove that they can be used to immobilize ß-trypsin at the surface.
Asunto(s)
Miniproteínas Nodales de Cistina/química , Proteínas Inmovilizadas/química , Hidrocarburos Aromáticos con Puentes/química , Miniproteínas Nodales de Cistina/farmacología , Imidazoles/química , Proteínas Inmovilizadas/farmacología , Modelos Moleculares , Estructura Secundaria de Proteína , Propiedades de Superficie , Inhibidores de Tripsina/química , Inhibidores de Tripsina/farmacologíaRESUMEN
Scorpion venoms include several compounds with different pharmacological activities. Within these compounds, toxins affecting ion channels are among the most studied. They are all peptides that have been classified based on their 3D structure, chain size and function. Usually, they show a spatial arrangement characterized by the presence of a cysteine-stabilized alpha beta motif; most of them affect Na(+) and K(+) ion-channels. These features have been revised in several occasions before, but a complete phylogenetic analysis of the disulfide containing peptides is not been done. In the present contribution, two databases (Pfam and InterPro) including more than 800 toxins from different scorpions were analyzed. Pfam database included toxins from several organisms other than scorpions such as insects and plants, while InterPro included only scorpion toxins. Our results suggest that Na(+) toxins have evolved independently from those of K(+) toxins no matter the length of the peptidic chains. These preliminary results suggest that current classification needs a more detailed revision, in order to have better characterized toxin families, so the new peptides obtained from transcriptomic analyses would be properly classified.
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Miniproteínas Nodales de Cistina/química , Evolución Molecular , Venenos de Escorpión/química , Teorema de Bayes , Bases de Datos de Compuestos Químicos , Filogenia , Estructura Terciaria de Proteína , Venenos de Escorpión/clasificaciónRESUMEN
For a long time the presence of knots in a protein structure was not admitted. However, the existence of proteins with various types of knots has now been proven. The functional significance of knotted topology remains unclear despite numerous assumptions. Studing the structure of knots in proteins and their impact on the acquisition of native structure of proteins is important for the understanding of protein folding as a whole. We review the types of knots in the proteins discovered to date, including trefoil knot, figure-of-eight knot, and more complex knots with 5 and 6 crossings of polypeptide chain. We survey the folding of knotted proteins as well.
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Proteínas Bacterianas/química , Miniproteínas Nodales de Cistina/química , Fitocromo/química , Proteínas de Plantas/química , ARNt Metiltransferasas/química , Animales , Humanos , Modelos Moleculares , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
Cystine knot α-amylase inhibitors belong to a knottin family of peptidyl inhibitors of 30-32 residues and contain two to four prolines. Thus far, only four members of the group of cystine knot α-amylase inhibitors have been characterized. Herein, the discovery and characterization of five cystine knot α-amylase inhibitors, allotides C1-C5 (Ac1-Ac5) (1-5), from the medicinal plant Allamanda cathartica are reported using both proteomic and genomic methods. Proteomic analysis showed that 1-5 are 30 amino acids in length with three or four proline residues. NMR determination of 4 revealed that it has two cis- and one trans-proline residues and adopts two equally populated conformations in solution. Determination of disulfide connectivity of 2 by differential S-reduction and S-alkylation provided clues of its unfolding process. Genomic analysis showed that allotide precursors contain a three-domain arrangement commonly found in plant cystine knot peptides with conserved residues flanking the processing sites of the mature allotide domain. This work expands the number of known cystine knot α-amylase inhibitors and furthers the understanding of both the structural and biological diversity of this type of knottin family.