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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 Nat Prod ; 2020 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-32437150

RESUMO

Viola is the largest genus in the Violaceae plant family and is known for its ubiquitous natural production of cyclotides. Many Viola species are used as medicinal herbs across Asia and are often consumed by humans in teas for the treatment of diseases, including ulcers and asthma. Previous studies reported the isolation of cyclotides from Viola species in many countries in the hope of discovering novel compounds with anti-cancer activities; however, Viola species from Vietnam have not been investigated to date. Here, the discovery of cyclotides from three Viola species (V. arcuata, V. tonkinensis, and V. austrosinensis) collected in the northern mountainous region of Vietnam is reported. Ten cyclotides were isolated from these three Viola species: four are novel and six were previously reported to be expressed in other plants. The structures of three of the new bracelet cyclotides are similar to that of cycloviolacin O2. Because cycloviolacin O2 has previously been shown to have potent activity against a wide range of cancer cell lines including HeLa (human cervical cancer cells) and PC-3 (human prostate cancer cells), the cancer cytotoxicity of the cyclotides isolated from V. arcuata was assessed. All tested cyclotides were cytotoxic against cancer cells, albeit to varying degrees. The sequences discovered in this study significantly expand the understanding of cyclotide diversity, especially in comparison with other cyclotides found in plants from the Asian region.

3.
Artigo em Inglês | MEDLINE | ID: mdl-32270580

RESUMO

Ruthenium-catalysed azide-alkyne cycloaddition (RuAAC) provides access to 1,5-disubstituted 1,2,3-triazole motifs in peptide engineering applications. However, investigation of this motif as a disulfide mimetic in cyclic peptides has been limited, and the structural consequences remain to be studied. We report synthetic strategies to install various triazole linkages into cyclic peptides through backbone cyclisation and RuAAC cross-linking reactions. These linkages were evaluated in four serine protease inhibitors based on sunflower trypsin inhibitor-1. NMR and X-ray crystallography revealed exceptional consensus of bridging distance and backbone conformations (RMSD<0.5 Å) of the triazole linkages compared to the parent disulfide molecules. The triazole-bridged peptides also displayed superior half-lives in liver S9 stability assays compared to disulfide-bridged peptides. This work establishes a foundation for the application of 1,5-disubstituted 1,2,3-triazoles as disulfide mimetics.

4.
ACS Chem Biol ; 15(6): 1650-1661, 2020 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-32315152

RESUMO

Cyclotides are macrocyclic peptides with exceptionally stable structures and have been reported to penetrate cells, making them promising scaffolds for the delivery of inhibitory peptides to target intracellular proteins. However, their cellular uptake and cytosolic localization have been poorly understood until now, which has limited their therapeutic potential. In this study, the recently developed chloroalkane penetration assay was combined with established assays to characterize the cellular uptake and cytosolic delivery of the prototypic cyclotide, kalata B1. We show that kalata B1 enters the cytosol at low efficiency. A structure-activity study of residues in loop 6 showed that some modifications, such as increasing cationic residue content, did not affect delivery efficiency, whereas others, including introducing a single hydrophobic amino acid, did significantly improve cytosolic delivery. Our results provide a foundation for the further development of a structurally unique class of scaffolds for the delivery of therapeutic cargoes into cells.

5.
Nat Commun ; 11(1): 1575, 2020 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-32221295

RESUMO

Asparaginyl endopeptidases (AEPs) catalyze the key backbone cyclization step during the biosynthesis of plant-derived cyclic peptides. Here, we report the identification of two AEPs from Momordica cochinchinensis and biochemically characterize MCoAEP2 that catalyzes the maturation of trypsin inhibitor cyclotides. Recombinantly produced MCoAEP2 catalyzes the backbone cyclization of a linear cyclotide precursor (MCoTI-II-NAL) with a kcat/Km of 620 mM-1 s-1, making it one of the fastest cyclases reported to date. We show that MCoAEP2 can mediate both the N-terminal excision and C-terminal cyclization of cyclotide precursors in vitro. The rate of cyclization/hydrolysis is primarily influenced by varying pH, which could potentially control the succession of AEP-mediated processing events in vivo. Furthermore, MCoAEP2 efficiently catalyzes the backbone cyclization of an engineered MCoTI-II analog with anti-angiogenic activity. MCoAEP2 provides enhanced synthetic access to structures previously inaccessible by direct chemistry approaches and enables the wider application of trypsin inhibitor cyclotides in biotechnology applications.

6.
ACS Chem Biol ; 15(4): 962-969, 2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32203656

RESUMO

Cyclotides are a class of cyclic disulfide-rich peptides found in plants that have been adopted as a molecular scaffold for pharmaceutical applications due to their inherent stability and ability to penetrate cell membranes. For research purposes, they are usually produced and cyclized synthetically, but there are concerns around the cost and environmental impact of large-scale chemical synthesis. One strategy to improve this is to combine a recombinant production system with native enzyme-mediated cyclization. Asparaginyl endopeptidases (AEPs) are enzymes that can act as peptide ligases in certain plants to facilitate cyclotide maturation. One of these ligases, OaAEP1b, originates from the cyclotide-producing plant, Oldenlandia affinis, and can be produced recombinantly for use in vitro as an alternative to chemical cyclization of recombinant substrates. However, not all engineered cyclotides are compatible with AEP-mediated cyclization because new pharmaceutical epitopes often replace the most flexible region of the peptide, where the native cyclization site is located. Here we redesign a popular cyclotide grafting scaffold, MCoTI-II, to incorporate an AEP cyclization site located away from the usual grafting region. We demonstrate the incorporation of a bioactive peptide sequence in the most flexible region of MCoTI-II while maintaining AEP compatibility, where the two were previously mutually exclusive. We anticipate that our AEP-compatible scaffold, based on the most popular cyclotide for pharmaceutical applications, will be useful in designing bioactive cyclotides that are compatible with AEP-mediated cyclization and will therefore open up the possibility of larger scale enzyme-mediated production of recombinant or synthetic cyclotides alike.

7.
J Biol Chem ; 295(15): 5067-5080, 2020 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-32139508

RESUMO

Huwentoxin-IV (HwTx-IV) is a gating modifier peptide toxin from spiders that has weak affinity for the lipid bilayer. As some gating modifier toxins have affinity for model lipid bilayers, a tripartite relationship among gating modifier toxins, voltage-gated ion channels, and the lipid membrane surrounding the channels has been proposed. We previously designed an HwTx-IV analogue (gHwTx-IV) with reduced negative charge and increased hydrophobic surface profile, which displays increased lipid bilayer affinity and in vitro activity at the voltage-gated sodium channel subtype 1.7 (NaV1.7), a channel targeted in pain management. Here, we show that replacements of the positively-charged residues that contribute to the activity of the peptide can improve gHwTx-IV's potency and selectivity for NaV1.7. Using HwTx-IV, gHwTx-IV, [R26A]gHwTx-IV, [K27A]gHwTx-IV, and [R29A]gHwTx-IV variants, we examined their potency and selectivity at human NaV1.7 and their affinity for the lipid bilayer. [R26A]gHwTx-IV consistently displayed the most improved potency and selectivity for NaV1.7, examined alongside off-target NaVs, compared with HwTx-IV and gHwTx-IV. The lipid affinity of each of the three novel analogues was weaker than that of gHwTx-IV, but stronger than that of HwTx-IV, suggesting a possible relationship between in vitro potency at NaV1.7 and affinity for lipid bilayers. In a murine NaV1.7 engagement model, [R26A]gHwTx-IV exhibited an efficacy comparable with that of native HwTx-IV. In summary, this study reports the development of an HwTx-IV analogue with improved in vitro selectivity for the pain target NaV1.7 and with an in vivo efficacy similar to that of native HwTx-IV.

8.
J Med Chem ; 63(7): 3475-3484, 2020 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-32003561

RESUMO

Tachyplesin I (TPI) is a cationic ß-hairpin antimicrobial peptide with broad-spectrum, potent antimicrobial activity. In this study, the all d-amino acid analogue of TPI (TPAD) was synthesized, and its structure and activity were determined. TPAD has comparable antibacterial activity to TPI on 14 bacterial strains, including four drug-resistant bacteria. Importantly, TPAD has significantly improved stability against enzymatic degradation and decreased hemolytic activity compared to TPI, indicating that it has better therapeutic potential. The induction of bacterial resistance using low concentrations of TPAD resulted in the activation of the QseC/B two-component system. Deletion of this system resulted in at least five-fold improvement of TPAD activity, and the combined use of TPAD with LED209, a QseC/B inhibitor, significantly enhanced the bactericidal effect against three classes of multidrug-resistant bacteria.

9.
J Med Chem ; 63(4): 1564-1575, 2020 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-31986036

RESUMO

αO-conotoxin GeXIVA from Conus generalis is a potent antagonist of the α9α10 nAChR and analgesic in animal models of pain. This peptide has two disulfide bond cross-links, and the bead and ribbon isomers have similar inhibitory activity against α9α10 nAChRs. We synthesized 12 disulfide-deficient analogues of bead GeXIVA, and all remained potent inhibitors of α9α10 nAChR. The most potent disulfide-deficient analogue displayed IC50 values of 6 and 33 nM at rat and human α9α10 nAChRs, respectively, representing less than a 2-fold increase compared with bead GeXIVA. The disulfide-deficient analogs and parent peptides also do not have a well-defined structure according to NMR spectroscopy. Molecular simulations suggest that the disulfide bonds and termini of GeXIVA do not establish stable interactions with the receptor. Overall, this study proposes that the structure of the analgesic peptide GeXIVA could be simplified through disulfide bond deletions and potentially termini truncations.

10.
Mar Drugs ; 18(1)2020 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-31940927

RESUMO

This study reports the isolation of two novel cysteine-rich antibacterial peptides, turgencin A and turgencin B, along with their oxidized derivatives, from the Arctic marine colonial ascidian Synoicum turgens. The peptides are post-translationally modified, containing six cysteines with an unusual disulfide connectivity of Cys1-Cys6, Cys2-Cys5, and Cys3-Cys4 and an amidated C-terminus. Furthermore, the peptides contain methionine residues resulting in the isolation of peptides with different degrees of oxidation. The most potent peptide, turgencin AMox1 with one oxidized methionine, displayed antimicrobial activity against both Gram-negative and Gram-positive bacteria with a minimum inhibitory concentration (MIC) as low as 0.4 µM against selected bacterial strains. In addition, the peptide inhibited the growth of the melanoma cancer cell line A2058 (IC50 = 1.4 µM) and the human fibroblast cell line MRC-5 (IC50 = 4.8 µM). The results from this study show that natural peptides isolated from marine tunicates have the potential to be promising drug leads.

11.
J Med Chem ; 63(2): 816-826, 2020 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-31855419

RESUMO

Chymase is a serine protease that is predominantly expressed by mast cells and has key roles in immune defense and the cardiovascular system. This enzyme has also emerged as a therapeutic target for cardiovascular disease due to its ability to remodel cardiac tissue and generate angiotensin II. Here, we used the nature-derived cyclic peptide sunflower trypsin inhibitor-1 (SFTI-1) as a template for designing novel chymase inhibitors. The key binding contacts of SFTI-1 were optimized by combining a peptide substrate library screen with structure-based design, which yielded several variants with potent activity. The lead variant was further modified by replacing the P1 Tyr residue with para-substituted Phe derivatives, generating new inhibitors with improved potency (Ki = 1.8 nM) and higher selectivity over closely related enzymes. Several variants were shown to block angiotensin I cleavage in vitro, highlighting their potential for further development and future evaluation as pharmaceutical leads.

12.
Chem Rev ; 119(24): 12375-12421, 2019 12 26.
Artigo em Inglês | MEDLINE | ID: mdl-31829013

RESUMO

This Review explores the class of plant-derived macrocyclic peptides called cyclotides. We include an account of their discovery, characterization, and distribution in the plant kingdom as well as a detailed analysis of their sequences and structures, biosynthesis and chemical synthesis, biological functions, and applications. These macrocyclic peptides are around 30 amino acids in size and are characterized by their head-to-tail cyclic backbone and cystine knot motif, which render them to be exceptionally stable, with resistance to thermal or enzymatic degradation. Routes to their chemical synthesis have been developed over the past two decades, and this capability has facilitated a wide range of mutagenesis and structure-activity relationship studies. In turn, these studies have both led to an increased understanding of their mechanisms of action as well as facilitated a range of applications in agriculture and medicine, as ecofriendly crop protection agents, and as drug leads or scaffolds for pharmaceutical design. Our overall objective in this Review is to provide readers with a comprehensive overview of cyclotides that we hope will stimulate further work on this fascinating family of peptides.

13.
Artigo em Inglês | MEDLINE | ID: mdl-31867866

RESUMO

Disulfide bonds typically introduce conformational constraints into peptides and proteins, conferring improved biopharmaceutical properties and greater therapeutic potential. In our opinion, disulfide-rich microdomains from proteins are potentially a rich and under-explored source of drug leads. A survey of the UniProt protein database shows that these domains are widely distributed throughout the plant and animal kingdoms, with the EGF-like domain being the most abundant of these domains. EGF-like domains exhibit large diversity in their disulfide bond topologies and calcium binding modes, which we classify in detail here. We found that many EGF-like domains are associated with disease phenotypes, and the interactions they mediate are potential therapeutic targets. Indeed, EGF-based therapeutic leads have been identified, and we further propose that these domains can be optimized to expand their therapeutic potential using chemical design strategies. This Review highlights the potential of disulfide-rich microdomains as future peptide therapeutics.

14.
ACS Chem Biol ; 14(12): 2895-2908, 2019 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-31714739

RESUMO

Tachyplesin-I (TI) is a host defense peptide from the horseshoe crab Tachypleus tridentatus that has outstanding potential as an anticancer therapeutic lead. Backbone cyclized TI (cTI) has similar anticancer properties to TI but has higher stability and lower hemolytic activity. We designed and synthesized cTI analogues to further improve anticancer potential and investigated structure-activity relationships based on peptide-membrane interactions, cellular uptake, and anticancer activity. The membrane-binding affinity and cytotoxic activity of cTI were found to be highly dependent on peptide hydrophobicity and charge. We describe two analogues with increased selectivity toward melanoma cells and one analogue with the ability to enter cells with high efficacy and low toxicity. Overall, the structure-activity relationship study shows that cTI can be developed as a membrane-active antimelanoma lead, or be employed as a cell penetrating peptide scaffold that can target and enter cells without damaging their integrity.


Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Antineoplásicos/farmacologia , Peptídeos Penetradores de Células/farmacologia , Proteínas de Ligação a DNA/farmacologia , Caranguejos Ferradura/química , Peptídeos Cíclicos/farmacologia , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos/química , Antineoplásicos/química , Linhagem Celular , Peptídeos Penetradores de Células/química , Proteínas de Ligação a DNA/química , Humanos , Peptídeos Cíclicos/química
15.
Toxicon ; 172: 33-44, 2019 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-31682883

RESUMO

Cyclotides are a plant-derived family of peptides that comprise approximately 30 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.


Assuntos
Ciclotídeos/química , Magnoliopsida/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Ciclotídeos/farmacologia , Ciclotídeos/toxicidade , Insetos/efeitos dos fármacos , Toxinas Biológicas/química , Toxinas Biológicas/toxicidade
16.
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.

17.
Molecules ; 24(20)2019 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-31627265

RESUMO

Grb7 is an adapter protein, overexpressed in HER2+ve breast and other cancers, and identified as a therapeutic target. Grb7 promotes both proliferative and migratory cellular pathways through interaction of its SH2 domain with upstream binding partners including HER2, SHC, and FAK. Here we present the evaluation of a series of monocyclic and bicyclic peptide inhibitors that have been developed to specifically and potently target the Grb7 SH2-domain. All peptides tested were found to inhibit signaling in both ERK and AKT pathways in SKBR-3 and MDA-MB-231 cell lines. Proliferation, migration, and invasion assays revealed, however, that the second-generation bicyclic peptides were not more bioactive than the first generation G7-18NATE peptide, despite their higher in vitro affinity for the target. This was found not to be due to steric hindrance by the cell-permeability tag, as ascertained by ITC, but to differences in the ability of the bicyclic peptides to interact with and penetrate cellular membranes, as determined using SPR and mass spectrometry. These studies reveal that just small differences to amino acid composition can greatly impact the effectiveness of peptide inhibitors to their intracellular target and demonstrate that G7-18NATE remains the most effective peptide inhibitor of Grb7 developed to date.


Assuntos
Antineoplásicos/farmacologia , Células Epiteliais/efeitos dos fármacos , Proteína Adaptadora GRB7/antagonistas & inibidores , Regulação Neoplásica da Expressão Gênica , Peptídeos Cíclicos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Sequência de Aminoácidos , Antineoplásicos/síntese química , Sítios de Ligação , Linhagem Celular , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Proteína Adaptadora GRB7/genética , Proteína Adaptadora GRB7/metabolismo , Humanos , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Peptídeos Cíclicos/síntese química , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Fosfatidilserinas/química , Fosfatidilserinas/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/genética , Relação Estrutura-Atividade , Domínios de Homologia de src/efeitos dos fármacos
18.
Chem Rev ; 119(21): 11510-11549, 2019 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-31633928

RESUMO

The venom of the marine predatory cone snails (genus Conus) has evolved for prey capture and defense, providing the basis for survival and rapid diversification of the now estimated 750+ species. A typical Conus venom contains hundreds to thousands of bioactive peptides known as conotoxins. These mostly disulfide-rich and well-structured peptides act on a wide range of targets such as ion channels, G protein-coupled receptors, transporters, and enzymes. Conotoxins are of interest to neuroscientists as well as drug developers due to their exquisite potency and selectivity, not just against prey but also mammalian targets, thereby providing a rich source of molecular probes and therapeutic leads. The rise of integrated venomics has accelerated conotoxin discovery with now well over 10,000 conotoxin sequences published. However, their structural and pharmacological characterization lags considerably behind. In this review, we highlight the diversity of new conotoxins uncovered since 2014, their three-dimensional structures and folds, novel chemical approaches to their syntheses, and their value as pharmacological tools to unravel complex biology. Additionally, we discuss challenges and future directions for the field.

19.
J Am Chem Soc ; 141(43): 17388-17393, 2019 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-31573802

RESUMO

Protein ligases of defined substrate specificity are versatile tools for protein engineering. Upon completion of the reaction, the products of currently reported protein ligases contain the amino acid sequence that is recognized by that same ligase, resulting in repeated cycles of ligation and hydrolysis as competing reactions. Thus, previous efforts to sequentially label proteins at distinct positions required ligases of orthogonal specificity. A recombinant Oldenlandia affinis asparaginyl endopeptidase, OaAEP1, is promiscuous for incoming nucleophiles. This promiscuity enabled us to define a nucleophile composed of natural amino acids that is ligated efficiently to the substrate yet yields a product that is poorly recognized by OaAEP1. Proteins modified with an efficient recognition module could be readily modified to yield a defined product bearing a cleavage-resistant motif, whereas proteins containing this inferior recognition motif remained essentially unmodified. We demonstrate the versatility of the N- or C-terminal protein modifications obtainable with this approach and modify the N- and C-termini of a single substrate protein in a sequential, site-specific manner in excellent yield.

20.
ChemMedChem ; 14(19): 1710-1716, 2019 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-31444979

RESUMO

Naturally occurring constrained peptides are frequently used as scaffolds for bioactive peptide grating due to their high stability. Here, we used in silico methods to design several constrained peptides comprising a scorpion toxin scaffold, a MDM2 binding epitope, and a cluster of positively charged residues. The designed peptides displayed varied binding affinity to MDM2 despite differing by only one or two residues. One of the peptides, SC426, had nanomolar binding affinity (KD =6.6±2.6 nm) to MDM2, and exhibited stronger inhibitory activity on the proliferation of HCT116 cells (p53-wild type) and SW480 cells (p53-mutant) than that of nutlin-3a. Binding mode analysis of the designed peptide at MDM2 suggests that the conserved "FWL" epitope was buried in the hydrophobic binding pocket, and the residues located at the periphery of the binding site contributed to the high binding affinity of SC426. Overall, in silico design of miniproteins with therapeutic potential through epitope grafting to the naturally occurring constrained peptide is an effective strategy.

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