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1.
J Biol Chem ; 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32414842

RESUMO

Cyclotides are plant-derived peptides characterized by a ~30-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 one 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, 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.

2.
J Am Chem Soc ; 141(51): 20460-20469, 2019 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-31765148

RESUMO

Peptides with pharmaceutical activities are attractive drug leads, and knowledge of their mode-of-action is essential for translation into the clinic. Comparison of native and enantiomeric peptides has long been used as a powerful approach to discriminate membrane- or receptor-mediated modes-of-action on the basis of the assumption that interactions with cell membranes are independent of peptide chirality. Here, we revisit this paradigm with the cyclotide kalata B1, a drug scaffold with intrinsic membrane-binding activity whose enantiomer is less potent than native peptide. To investigate this chirality dependence, we compared peptide-lipid binding using mirror image model membranes. We synthesized phospholipids with non-natural chirality and demonstrate that native kalata B1 binds with higher affinity to phospholipids with chirality found in eukaryotic membranes. This study shows for the first time that the chiral environment of lipid bilayers can modulate the function of membrane-active peptides and challenges the view that peptide-lipid interactions are achiral.

3.
ACS Chem Biol ; 14(1): 118-130, 2019 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-30507158

RESUMO

Gating modifier toxins (GMTs) from spider venom can inhibit voltage gated sodium channels (NaVs) involved in pain signal transmission, including the NaV1.7 subtype. GMTs have a conserved amphipathic structure that allow them to interact with membranes and also with charged residues in regions of NaV that are exposed at the cell surface. ProTx-II and GpTx-1 are GMTs able to inhibit NaV1.7 with high potency, but they differ in their ability to bind to membranes and in their selectivity over other NaV subtypes. To explore these differences and gain detailed information on their membrane-binding ability and how this relates to potency and selectivity, we examined previously described NaV1.7 potent/selective GpTx-1 analogues and new ProTx-II analogues designed to reduce membrane binding and improve selectivity for NaV1.7. Our studies reveal that the number and type of hydrophobic residues as well as how they are presented at the surface determine the affinity of ProTx-II and GpTx-1 for membranes and that altering these residues can have dramatic effects on NaV inhibitory activity. We demonstrate that strong peptide-membrane interactions are not essential for inhibiting NaV1.7 and propose that hydrophobic interactions instead play an important role in positioning the GMT at the membrane surface proximal to exposed NaV residues, thereby affecting peptide-channel interactions. Our detailed structure-activity relationship study highlights the challenges of designing GMT-based molecules that simultaneously achieve high potency and selectivity for NaV1.7, as single mutations can induce local changes in GMT structure that can have a major impact on NaV-inhibitory activity.


Assuntos
Peptídeos/efeitos dos fármacos , Venenos de Aranha/farmacologia , Animais , Humanos , Peptídeos/química
4.
Cell Mol Life Sci ; 75(24): 4511-4524, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30109357

RESUMO

Sea anemone venoms have long been recognized as a rich source of peptides with interesting pharmacological and structural properties, but they still contain many uncharacterized bioactive compounds. Here we report the discovery, three-dimensional structure, activity, tissue localization, and putative function of a novel sea anemone peptide toxin that constitutes a new, sixth type of voltage-gated potassium channel (KV) toxin from sea anemones. Comprised of just 17 residues, κ-actitoxin-Ate1a (Ate1a) is the shortest sea anemone toxin reported to date, and it adopts a novel three-dimensional structure that we have named the Proline-Hinged Asymmetric ß-hairpin (PHAB) fold. Mass spectrometry imaging and bioassays suggest that Ate1a serves a primarily predatory function by immobilising prey, and we show this is achieved through inhibition of Shaker-type KV channels. Ate1a is encoded as a multi-domain precursor protein that yields multiple identical mature peptides, which likely evolved by multiple domain duplication events in an actinioidean ancestor. Despite this ancient evolutionary history, the PHAB-encoding gene family exhibits remarkable sequence conservation in the mature peptide domains. We demonstrate that this conservation is likely due to intra-gene concerted evolution, which has to our knowledge not previously been reported for toxin genes. We propose that the concerted evolution of toxin domains provides a hitherto unrecognised way to circumvent the effects of the costly evolutionary arms race considered to drive toxin gene evolution by ensuring efficient secretion of ecologically important predatory toxins.


Assuntos
Venenos de Cnidários/química , Peptídeos/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Anêmonas-do-Mar/química , Sequência de Aminoácidos , Animais , Venenos de Cnidários/genética , Venenos de Cnidários/metabolismo , Evolução Molecular , Modelos Moleculares , Peptídeos/genética , Peptídeos/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Conformação Proteica , Dobramento de Proteína , Anêmonas-do-Mar/genética , Anêmonas-do-Mar/metabolismo , Transcriptoma
5.
Cell Chem Biol ; 25(9): 1140-1150.e5, 2018 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-30033131

RESUMO

Malaria is a serious threat to human health and additional classes of antimalarial drugs are greatly needed. The human defense protein, platelet factor 4 (PF4), has intrinsic antiplasmodial activity but also undesirable chemokine properties. We engineered a peptide containing the isolated PF4 antiplasmodial domain, which through cyclization, retained the critical structure of the parent protein. The peptide, cPF4PD, killed cultured blood-stage Plasmodium falciparum with low micromolar potency by specific disruption of the parasite digestive vacuole. Its mechanism of action involved selective penetration and accumulation inside the intraerythrocytic parasite without damaging the host cell or parasite membranes; it did not accumulate in uninfected cells. This selective activity was accounted for by observations of the peptide's specific binding and penetration of membranes with exposed negatively charged phospholipid headgroups. Our findings highlight the tremendous potential of the cPF4PD scaffold for developing antimalarial peptide drugs with a distinct and selective mechanism of action.


Assuntos
Antimaláricos/química , Antimaláricos/farmacologia , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Fator Plaquetário 4/química , Fator Plaquetário 4/farmacologia , Adulto , Desenho de Fármacos , Eritrócitos/parasitologia , Feminino , Humanos , Malária Falciparum/tratamento farmacológico , Malária Falciparum/parasitologia , Masculino , Pessoa de Meia-Idade , Modelos Moleculares
6.
J Biol Chem ; 293(23): 9041-9052, 2018 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-29703751

RESUMO

Gating modifier toxins (GMTs) are venom-derived peptides isolated from spiders and other venomous creatures and modulate activity of disease-relevant voltage-gated ion channels and are therefore being pursued as therapeutic leads. The amphipathic surface profile of GMTs has prompted the proposal that some GMTs simultaneously bind to the cell membrane and voltage-gated ion channels in a trimolecular complex. Here, we examined whether there is a relationship among spider GMT amphipathicity, membrane binding, and potency or selectivity for voltage-gated sodium (NaV) channels. We used NMR spectroscopy and in silico calculations to examine the structures and physicochemical properties of a panel of nine GMTs and deployed surface plasmon resonance to measure GMT affinity for lipids putatively found in proximity to NaV channels. Electrophysiology was used to quantify GMT activity on NaV1.7, an ion channel linked to chronic pain. Selectivity of the peptides was further examined against a panel of NaV channel subtypes. We show that GMTs adsorb to the outer leaflet of anionic lipid bilayers through electrostatic interactions. We did not observe a direct correlation between GMT amphipathicity and affinity for lipid bilayers. Furthermore, GMT-lipid bilayer interactions did not correlate with potency or selectivity for NaVs. We therefore propose that increased membrane binding is unlikely to improve subtype selectivity and that the conserved amphipathic GMT surface profile is an adaptation that facilitates simultaneous modulation of multiple NaVs.


Assuntos
Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Venenos de Aranha/farmacologia , Toxinas Biológicas/farmacologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/metabolismo , Proteínas de Artrópodes/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células HEK293 , Humanos , Bicamadas Lipídicas/metabolismo , Modelos Moleculares , Venenos de Aranha/química , Venenos de Aranha/metabolismo , Aranhas/química , Toxinas Biológicas/química , Toxinas Biológicas/metabolismo , Bloqueadores do Canal de Sódio Disparado por Voltagem/química , Bloqueadores do Canal de Sódio Disparado por Voltagem/metabolismo , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia
7.
Cell Chem Biol ; 25(4): 380-391.e5, 2018 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-29396290

RESUMO

Resistance to the last-resort antibiotic colistin is now widespread and new therapeutics are urgently required. We report the first in toto chemical synthesis and pre-clinical evaluation of octapeptins, a class of lipopeptides structurally related to colistin. The octapeptin biosynthetic cluster consisted of three non-ribosomal peptide synthetases (OctA, OctB, and OctC) that produced an amphiphilic antibiotic, octapeptin C4, which was shown to bind to and depolarize membranes. While active against multi-drug resistant (MDR) strains in vitro, octapeptin C4 displayed poor in vivo efficacy, most likely due to high plasma protein binding. Nuclear magnetic resonance solution structures, empirical structure-activity and structure-toxicity models were used to design synthetic octapeptins active against MDR and extensively drug-resistant (XDR) bacteria. The scaffold was then subtly altered to reduce plasma protein binding, while maintaining activity against MDR and XDR bacteria. In vivo efficacy was demonstrated in a murine bacteremia model with a colistin-resistant P. aeruginosa clinical isolate.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , Infecções por Bactérias Gram-Negativas/tratamento farmacológico , Lipopeptídeos/química , Lipopeptídeos/farmacologia , Animais , Antibacterianos/efeitos adversos , Antibacterianos/uso terapêutico , Modelos Animais de Doenças , Farmacorresistência Bacteriana , Feminino , Humanos , Lipopeptídeos/efeitos adversos , Lipopeptídeos/uso terapêutico , Camundongos , Modelos Moleculares , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/efeitos dos fármacos
8.
J Biol Chem ; 293(5): 1536-1549, 2018 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-29255091

RESUMO

Crotalicidin (Ctn), a cathelicidin-related peptide from the venom of a South American rattlesnake, possesses potent antimicrobial, antitumor, and antifungal properties. Previously, we have shown that its C-terminal fragment, Ctn(15-34), retains the antimicrobial and antitumor activities but is less toxic to healthy cells and has improved serum stability. Here, we investigated the mechanisms of action of Ctn and Ctn(15-34) against Gram-negative bacteria. Both peptides were bactericidal, killing ∼90% of Escherichia coli and Pseudomonas aeruginosa cells within 90-120 and 5-30 min, respectively. Studies of ζ potential at the bacterial cell membrane suggested that both peptides accumulate at and neutralize negative charges on the bacterial surface. Flow cytometry experiments confirmed that both peptides permeabilize the bacterial cell membrane but suggested slightly different mechanisms of action. Ctn(15-34) permeabilized the membrane immediately upon addition to the cells, whereas Ctn had a lag phase before inducing membrane damage and exhibited more complex cell-killing activity, probably because of two different modes of membrane permeabilization. Using surface plasmon resonance and leakage assays with model vesicles, we confirmed that Ctn(15-34) binds to and disrupts lipid membranes and also observed that Ctn(15-34) has a preference for vesicles that mimic bacterial or tumor cell membranes. Atomic force microscopy visualized the effect of these peptides on bacterial cells, and confocal microscopy confirmed their localization on the bacterial surface. Our studies shed light onto the antimicrobial mechanisms of Ctn and Ctn(15-34), suggesting Ctn(15-34) as a promising lead for development as an antibacterial/antitumor agent.


Assuntos
Antibacterianos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Membrana Celular , Venenos de Crotalídeos , Crotalus , Escherichia coli , Fragmentos de Peptídeos , Pseudomonas aeruginosa , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Membrana Celular/química , Membrana Celular/metabolismo , Venenos de Crotalídeos/química , Venenos de Crotalídeos/farmacologia , Escherichia coli/química , Escherichia coli/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/metabolismo , Ressonância de Plasmônio de Superfície
9.
Langmuir ; 33(26): 6630-6637, 2017 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-28605904

RESUMO

Cyclotides are cyclic disulfide-rich peptides that are chemically and thermally stable and possess pharmaceutical and insecticidal properties. The activities reported for cyclotides correlate with their ability to target phosphatidylethanolamine (PE)-phospholipids and disrupt cell membranes. However, the mechanism by which this disruption occurs remains unclear. In the current study we examine the effect of the prototypic cyclotides, kalata B1 (kB1) and kalata B2 (kB2), on tethered lipid bilayer membranes (tBLMs) using swept frequency electrical impedance spectroscopy. We confirmed that kB1 and kB2 bind to bilayers only if they contain PE-phospholipids. We hypothesize that the increase in membrane conduction and capacitance observed upon addition of kB1 or kB2 is unlikely to result from ion channel like pores but is consistent with the formation of lipidic toroidal pores. This hypothesis is supported by the concentration dependence of effects of kB1 and kB2 being suggestive of a critical micelle concentration event rather than a progressive increase in conduction arising from increased channel insertion. Additionally, conduction behavior is readily reversible when the peptide is rinsed from the bilayer. Our results support a mechanism by which kB1 and kB2 bind to and disrupt PE-containing membranes by decreasing the overall membrane critical packing parameter, as would a surfactant, which then opens or increases the size of existing membrane defects. The cyclotides need not participate directly in the conductive pore but might exert their effect indirectly through altering membrane packing constraints and inducing purely lipidic conductive pores.


Assuntos
Tensoativos/química , Sequência de Aminoácidos , Ciclotídeos , Bicamadas Lipídicas
11.
Biopolymers ; 108(5)2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28459137

RESUMO

Chlorotoxin (CTX), a disulfide-rich peptide from the scorpion Leiurus quinquestriatus, has several promising biopharmaceutical properties, including preferential affinity for certain cancer cells, high serum stability, and cell penetration. These properties underpin its potential for use as a drug design scaffold, especially for the treatment of cancer; indeed, several analogs of CTX have reached clinical trials. Here, we focus on its ability to internalize into cells-a trait associated with a privileged subclass of peptides called cell-penetrating peptides-and whether it can be improved through conservative substitutions. Mutants of CTX were made using solid-phase peptide synthesis and internalization into human cervical carcinoma (HeLa) cells was monitored by fluorescence and confocal microscopy. CTX_M1 (ie, [K15R/K23R]CTX) and CTX_M2 (ie, [K15R/K23R/Y29W]CTX) mutants showed at least a twofold improvement in uptake compared to CTX. We further showed that these mutants internalize into HeLa cells largely via an energy-dependent mechanism. Importantly, the mutants have high stability, remaining intact in serum for over 24 h; thus, retaining the characteristic stability of their parent peptide. Overall, we have shown that simple conservative substitutions can enhance the cellular uptake of CTX, suggesting that such type of mutations might be useful for improving uptake of other peptide toxins.


Assuntos
Venenos de Escorpião/metabolismo , Sequência de Aminoácidos , Animais , Arginina/química , Membrana Celular/química , Membrana Celular/metabolismo , Dissulfetos/química , Espectroscopia de Ressonância de Spin Eletrônica , Células HeLa , Humanos , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Lisina/química , Microscopia Confocal , Mutagênese , Estabilidade Proteica , Venenos de Escorpião/síntese química , Venenos de Escorpião/genética , Escorpiões/metabolismo , Alinhamento de Sequência
12.
Biophys J ; 112(4): 630-642, 2017 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-28256223

RESUMO

Cyclotides are ultra-stable cyclic disulfide-rich peptides from plants. Their biophysical effects and medically interesting activities are related to their membrane-binding properties, with particularly high affinity for phosphatidylethanolamine lipids. In this study we were interested in understanding the molecular details of cyclotide-membrane interactions, specifically with regard to the spatial orientation of the cyclotide kalata B1 from Oldenlandia affinis when embedded in a lipid bilayer. Our experimental approach was based on the use of solid-state 19F-NMR of oriented bilayers in conjunction with the conformationally restricted amino acid L-3-(trifluoromethyl)bicyclopent-[1.1.1]-1-ylglycine as an orientation-sensitive 19F-NMR probe. Its rigid connection to the kalata B1 backbone scaffold, together with the well-defined structure of the cyclotide, allowed us to calculate the protein alignment in the membrane directly from the orientation-sensitive 19F-NMR signal. The hydrophobic and polar residues on the surface of kalata B1 form well-separated patches, endowing this cyclotide with a pronounced amphipathicity. The peptide orientation, as determined by NMR, showed that this amphipathic structure matches the polar/apolar interface of the lipid bilayer very well. A location in the amphiphilic headgroup region of the bilayer was supported by 15N-NMR of uniformly labeled protein, and confirmed using solid-state 31P- and 2H-NMR. 31P-NMR relaxation data indicated a change in lipid headgroup dynamics induced by kalata B1. Changes in the 2H-NMR order parameter profile of the acyl chains suggest membrane thinning, as typically observed for amphiphilic peptides embedded near the polar/apolar bilayer interface. Furthermore, from the 19F-NMR analysis two important charged residues, E7 and R28, were found to be positioned equatorially. The observed location thus would be favorable for the postulated binding of E7 to phosphatidylethanolamine lipid headgroups. Furthermore, it may be speculated that this pair of side chains could promote oligomerization of kalata B1 through electrostatic intermolecular contacts via their complementary charges.


Assuntos
Ciclotídeos/química , Ciclotídeos/metabolismo , Bicamadas Lipídicas/metabolismo , Sequência de Aminoácidos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Oldenlandia/metabolismo , Conformação Proteica
13.
Biopolymers ; 108(1)2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27487329

RESUMO

Cyclotides are plant-derived host defense peptides displaying exceptional stability due to their cyclic cystine knot comprising three intertwined disulfide bonds and a cyclic backbone. Their six conserved cysteine residues are separated by backbone loops with diverse sequences. Prototypical cyclotides from the Möbius (kalata B1) and trypsin inhibitor (MCoTI-II) subfamilies lack sequence homology with one another, but both are able to penetrate cells, apparently via different mechanisms. To delineate the influence of the sequences of the loops on the structure and cell internalization of these two cyclotide subfamilies, a series of Möbius/trypsin inhibitor loop-chimeras of kalata B1 and MCoTI-II were synthesized, and structurally and functionally characterized. NMR analysis showed that the structural fold of the majority of chimeric peptides was minimally affected by the loop substitutions. Substituting loops 3, 5, or 6 of MCoTI-II into the corresponding loops of kalata B1 attenuated its hemolytic and cytotoxic activities, and greatly reduced its cell-penetrating properties. On the other hand, replacing loops of MCoTI-II with the corresponding loops of kalata B1 did not introduce cytotoxicity into the chimeras. Loops 2, 3, and 4 of MCoTI-II were found to contribute little to cell-penetrating properties. Overall, this study provides valuable insights into the structural basis for the hemolytic, cytotoxic, and cell-penetrating properties of kalata B1 and MCoTI-II, which could be useful for future engineering of cyclotides to carry bioactive epitopes to intracellular targets.


Assuntos
Ciclotídeos/química , Proteínas de Plantas/química , Sequência de Aminoácidos , Sobrevivência Celular/efeitos dos fármacos , Cucurbitaceae/metabolismo , Ciclotídeos/síntese química , Ciclotídeos/toxicidade , Eritrócitos/citologia , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Células HeLa , Hemólise/efeitos dos fármacos , Humanos , Espectroscopia de Ressonância Magnética , Estrutura Terciária de Proteína
14.
Mech Ageing Dev ; 161(Pt B): 247-254, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27491841

RESUMO

The plant-derived decapeptide OSIP108 increases tolerance of yeast and human cells to apoptosis-inducing agents, such as copper and cisplatin. We performed a whole amino acid scan of OSIP108 and conducted structure-activity relationship studies on the induction of cisplatin tolerance (CT) in yeast. The use of cisplatin as apoptosis-inducing trigger in this study should be considered as a tool to better understand the survival-promoting nature of OSIP108 and not for purposes related to anti-cancer treatment. We found that charged residues (Arg, His, Lys, Glu or Asp) or a Pro on positions 4-7 improved OSIP108 activity by 10% or more. The variant OSIP108[G7P] induced the most pronounced tolerance to toxic concentrations of copper and cisplatin in yeast and/or HepG2 cells. Both OSIP108 and OSIP108[G7P] were shown to internalize equally into HeLa cells, but at a higher rate than the inactive OSIP108[E10A], suggesting that the peptides can internalize into cells and that OSIP108 activity is dependent on subsequent intracellular interactions. In conclusion, our studies demonstrated that tolerance/survival-promoting properties of OSIP108 can be significantly improved by single amino acid substitutions, and that these properties are dependent on (an) intracellular target(s), yet to be determined.


Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/farmacologia , Proteínas de Arabidopsis/farmacocinética , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/farmacocinética , Cisplatino/farmacologia , Farmacorresistência Fúngica/efeitos dos fármacos , Células HeLa , Células Hep G2 , Humanos , Saccharomyces cerevisiae/metabolismo
15.
J Biol Chem ; 291(33): 17049-65, 2016 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-27311819

RESUMO

ProTx-II is a disulfide-rich peptide toxin from tarantula venom able to inhibit the human voltage-gated sodium channel 1.7 (hNaV1.7), a channel reported to be involved in nociception, and thus it might have potential as a pain therapeutic. ProTx-II acts by binding to the membrane-embedded voltage sensor domain of hNaV1.7, but the precise peptide channel-binding site and the importance of membrane binding on the inhibitory activity of ProTx-II remain unknown. In this study, we examined the structure and membrane-binding properties of ProTx-II and several analogues using NMR spectroscopy, surface plasmon resonance, fluorescence spectroscopy, and molecular dynamics simulations. Our results show a direct correlation between ProTx-II membrane binding affinity and its potency as an hNaV1.7 channel inhibitor. The data support a model whereby a hydrophobic patch on the ProTx-II surface anchors the molecule at the cell surface in a position that optimizes interaction of the peptide with the binding site on the voltage sensor domain. This is the first study to demonstrate that binding of ProTx-II to the lipid membrane is directly linked to its potency as an hNaV1.7 channel inhibitor.


Assuntos
Bicamadas Lipídicas/química , Simulação de Dinâmica Molecular , Canal de Sódio Disparado por Voltagem NAV1.7/química , Venenos de Aranha/química , Sítios de Ligação , Humanos , Ressonância Magnética Nuclear Biomolecular
16.
Biopolymers ; 106(6): 853-863, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27287767

RESUMO

The transcription factor p53 has a tumor suppressor role in leading damaged cells to apoptosis. Its activity is regulated/inhibited in healthy cells by the proteins MDM2 and MDMX. Overexpression of MDM2 and/or MDMX in cancer cells inactivates p53, facilitating tumor development. A 12-mer dual inhibitor peptide (pDI) was previously reported to be able to target and inhibit MDMX:p53 and MDM2:p53 interactions with nanomolar potency in vitro. With the aim of improving its cellular inhibitory activity, we produced a series of constrained pDI analogs featuring lactam staples that stabilize the bioactive helical conformation and fused them with a cell-penetrating peptide to increase cytosol delivery. We compared pDI and its analogs on their inhibitory potency, toxicity, and ability to enter cancer cells. Overall, the results show that these analogs keep their nanomolar affinity for MDM2 and MDMX and are highly active against cancer cells. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 853-863, 2016.


Assuntos
Antineoplásicos , Peptídeos Penetradores de Células , Sistemas de Liberação de Medicamentos , Complexos Multiproteicos , Proteínas Nucleares , Proteínas Proto-Oncogênicas c-mdm2 , Proteínas Proto-Oncogênicas , Proteína Supressora de Tumor p53 , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Proteínas de Ciclo Celular , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/farmacocinética , Peptídeos Penetradores de Células/farmacologia , Células HeLa , Humanos , Complexos Multiproteicos/antagonistas & inibidores , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismo
17.
J Am Chem Soc ; 138(17): 5706-13, 2016 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-27064294

RESUMO

Enantiomeric forms of BTD-2, PG-1, and PM-1 were synthesized to delineate the structure and function of these ß-sheet antimicrobial peptides. Activity and lipid-binding assays confirm that these peptides act via a receptor-independent mechanism involving membrane interaction. The racemic crystal structure of BTD-2 solved at 1.45 Å revealed a novel oligomeric form of ß-sheet antimicrobial peptides within the unit cell: an antiparallel trimer, which we suggest might be related to its membrane-active form. The BTD-2 oligomer extends into a larger supramolecular state that spans the crystal lattice, featuring a steric-zipper motif that is common in structures of amyloid-forming peptides. The supramolecular structure of BTD-2 thus represents a new mode of fibril-like assembly not previously observed for antimicrobial peptides, providing structural evidence linking antimicrobial and amyloid peptides.


Assuntos
Amiloide/química , Anti-Infecciosos/química , Peptídeos/química , Dicroísmo Circular , Cristalografia por Raios X , Conformação Proteica , Ressonância de Plasmônio de Superfície
18.
J Biol Chem ; 291(22): 11829-42, 2016 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-27026701

RESUMO

The µO-conotoxins MrVIA, MrVIB, and MfVIA inhibit the voltage-gated sodium channel NaV1.8, a well described target for the treatment of pain; however, little is known about the residues or structural elements that define this activity. In this study, we determined the three-dimensional structure of MfVIA, examined its membrane binding properties, performed alanine-scanning mutagenesis, and identified residues important for its activity at human NaV1.8. A second round of mutations resulted in (E5K,E8K)MfVIA, a double mutant with greater positive surface charge and greater affinity for lipid membranes compared with MfVIA. This analogue had increased potency at NaV1.8 and was analgesic in the mouse formalin assay.


Assuntos
Analgésicos/farmacologia , Membrana Celular/metabolismo , Conotoxinas/farmacologia , Canal de Sódio Disparado por Voltagem NAV1.8/metabolismo , Dor/prevenção & controle , Sequência de Aminoácidos , Animais , Comportamento Animal/efeitos dos fármacos , Cristalografia por Raios X , Eletrofisiologia , Células HEK293 , Humanos , Lipossomos , Espectroscopia de Ressonância Magnética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Canal de Sódio Disparado por Voltagem NAV1.8/química , Canal de Sódio Disparado por Voltagem NAV1.8/genética , Dor/induzido quimicamente , Conformação Proteica , Homologia de Sequência de Aminoácidos
19.
Biochim Biophys Acta ; 1858(4): 872-82, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26850736

RESUMO

Many venom peptides are potent and selective inhibitors of voltage-gated ion channels, including channels that are validated therapeutic targets for treatment of a wide range of human diseases. However, the development of novel venom-peptide-based therapeutics requires an understanding of their mechanism of action. In the case of voltage-gated ion channels, venom peptides act either as pore blockers that bind to the extracellular side of the channel pore or gating modifiers that bind to one or more of the membrane-embedded voltage sensor domains. In the case of gating modifiers, it has been debated whether the peptide must partition into the membrane to reach its binding site. In this study, we used surface plasmon resonance, fluorescence spectroscopy and molecular dynamics to directly compare the lipid-binding properties of two gating modifiers (µ-TRTX-Hd1a and ProTx-I) and two pore blockers (ShK and KIIIA). Only ProTx-I was found to bind to model membranes. Our results provide further evidence that the ability to insert into the lipid bilayer is not a requirement to be a gating modifier. In addition, we characterised the surface of ProTx-I that mediates its interaction with neutral and anionic phospholipid membranes and show that it preferentially interacts with anionic lipids.


Assuntos
Membranas/efeitos dos fármacos , Peptídeos/química , Venenos de Aranha/química , Sítios de Ligação/efeitos dos fármacos , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Membranas/química , Peptídeos/toxicidade , Venenos de Aranha/toxicidade
20.
Biochemistry ; 55(2): 396-405, 2016 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-26685975

RESUMO

The SET protein is a promising drug target in cancer therapy, because of its ability to inhibit the function of the tumor suppressor gene protein phosphatase 2A (PP2A). COG peptides, derived from apolipoprotein E (apoE), are potent antagonists of SET; they induce cytotoxicity in cancer cells upon binding to intracellular SET and modulate the nuclear factor kappa B (NF-κB) signaling pathway. However, the therapeutic potential of COG peptides is limited, because of their poor proteolytic stability and low bioavailability. In this study, the COG peptide, COG1410, was stabilized by grafting it onto the ultrastable cyclic peptide scaffold, Momordica cochinchinensis trypsin inhibitor-II (MCoTI-II). The grafted MCoTI-II peptides were cytotoxic to a cancer cell line and showed high stability in human serum. The most potent grafted MCoTI-II peptide inhibited lipopolysaccharide (LPS)-mediated activation of NF-κB in murine macrophages. Overall, this study demonstrates the application of the MCoTI-II scaffold for the development of stable peptide drugs for cancer therapy.


Assuntos
Chaperonas de Histonas/antagonistas & inibidores , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Apolipoproteínas E/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Ciclotídeos/química , Ciclotídeos/farmacologia , Humanos , Imagem por Ressonância Magnética , NF-kappa B/metabolismo , Regiões Promotoras Genéticas/genética , Proteína Fosfatase 2/metabolismo
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