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1.
J Mol Model ; 26(9): 231, 2020 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-32789582

RESUMO

The complement system plays a major role in human immunity, but its abnormal activation can have severe pathological impacts. By mimicking a natural mechanism of complement regulation, the small peptide compstatin has proven to be a very promising complement inhibitor. Over the years, several compstatin analogs have been created, with improved inhibitory potency. A recent analog is being developed as a candidate drug against several pathological conditions, including COVID-19. However, the reasons behind its higher potency and increased binding affinity to complement proteins are not fully clear. This computational study highlights the mechanistic properties of several compstatin analogs, thus complementing previous experimental studies. We perform molecular dynamics simulations involving six analogs alone in solution and two complexes with compstatin bound to complement component 3. These simulations reveal that all the analogs we consider, except the original compstatin, naturally adopt a pre-bound conformation in solution. Interestingly, this set of analogs adopting a pre-bound conformation includes analogs that were not known to benefit from this behavior. We also show that the most recent compstatin analog (among those we consider) forms a stronger hydrogen bond network with its complement receptor than an earlier analog.


Assuntos
Antivirais/química , Complemento C3/antagonistas & inibidores , Peptídeos Cíclicos/química , Peptídeos Cíclicos/metabolismo , Antivirais/metabolismo , Complemento C3/metabolismo , Infecções por Coronavirus/tratamento farmacológico , Humanos , Ligação de Hidrogênio , Simulação de Dinâmica Molecular , Pandemias , Pneumonia Viral/tratamento farmacológico , Relação Estrutura-Atividade
2.
Nat Commun ; 11(1): 4027, 2020 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-32788676

RESUMO

Programmed cell death or apoptosis is a central biological process that is dysregulated in many diseases, including inflammatory conditions and cancer. The detection and quantification of apoptotic cells in vivo is hampered by the need for fixatives or washing steps for non-fluorogenic reagents, and by the low levels of free calcium in diseased tissues that restrict the use of annexins. In this manuscript, we report the rational design of a highly stable fluorogenic peptide (termed Apo-15) that selectively stains apoptotic cells in vitro and in vivo in a calcium-independent manner and under wash-free conditions. Furthermore, using a combination of chemical and biophysical methods, we identify phosphatidylserine as a molecular target of Apo-15. We demonstrate that Apo-15 can be used for the quantification and imaging of drug-induced apoptosis in preclinical mouse models, thus creating opportunities for assessing the in vivo efficacy of anti-inflammatory and anti-cancer therapeutics.


Assuntos
Apoptose , Imageamento Tridimensional , Peptídeos Cíclicos/farmacologia , Sequência de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Feminino , Humanos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Peptídeos Cíclicos/administração & dosagem , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/química , Fagocitose/efeitos dos fármacos , Fosfatidilserinas/metabolismo
3.
Int J Nanomedicine ; 15: 4021-4047, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32606662

RESUMO

Purpose: Periodontitis is a chronic inflammatory disease associated with microbial accumulation. The purpose of this study was to reuse the agricultural waste to produce cellulose nanofibers (CNF) and further modification of the CNF with κ-carrageenan oligosaccharides (CO) for drug delivery. In addition, this study is focused on the antimicrobial activity of surfactin-loaded CO-CNF towards periodontal pathogens. Materials and Methods: A chemo-mechanical method was used to extract the CNF and the modification was done by using CO. The studies were further proceeded by adding different quantities of surfactin [50 mg (50 SNPs), 100 mg (100 SNPs), 200 mg (200 SNPs)] into the carrier (CO-CNF). The obtained materials were characterized, and the antimicrobial activity of surfactin-loaded CO-CNF was evaluated. Results: The obtained average size of CNF and CO-CNF after ultrasonication was 263 nm and 330 nm, respectively. Microscopic studies suggested that the CNF has a short diameter with long length and CO became cross-linked to form as beads within the CNF network. The addition of CO improved the degradation temperature, crystallinity, and swelling property of CNF. The material has a controlled drug release, and the entrapment efficiency and loading capacity of the drug were 53.15 ± 2.36% and 36.72 ± 1.24%, respectively. It has antioxidant activity and inhibited the growth of periodontal pathogens such as Streptococcus mutans and Porphyromonas gingivalis by preventing the biofilm formation, reducing the metabolic activity, and promoting the oxidative stress. Conclusion: The study showed the successful extraction of CNF and modification with CO improved the physical parameters of the CNF. In addition, surfactin-loaded CO-CNF has potential antimicrobial activity against periodontal pathogens. The obtained biomaterial is economically valuable and has great potential for biomedical applications.


Assuntos
Carragenina/química , Celulose/química , Lipopeptídeos/química , Nanofibras/química , Peptídeos Cíclicos/química , Periodonto/microbiologia , Animais , Bactérias/metabolismo , Compostos de Bifenilo/química , Sobrevivência Celular , Difusão Dinâmica da Luz , Depuradores de Radicais Livres/química , Malondialdeído/metabolismo , Camundongos , Testes de Sensibilidade Microbiana , Nanofibras/ultraestrutura , Oligossacarídeos/química , Picratos/química , Células RAW 264.7 , Soja/química , Espectroscopia de Infravermelho com Transformada de Fourier
4.
J Chromatogr A ; 1623: 461198, 2020 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-32505287

RESUMO

Microcystins (MCs) and nodularin (NOD) are tumor promoters produced by cyanobacteria and present in surface water. In this work, a novel mesoporous metal-organic framework-5@chitosan (MOF-5@CS) material was synthesized and applied for the enrichment of MCs and NOD in water and fish samples. The mesoporous MOF-5@CS material was firstly synthesized via a one-step hydrothermal method, and the chitosan was combined with MOF-5 via chemical bonding assembly. As a new adsorbent, the as-synthesized material was found having a large specific surface area and good thermal stability. Under the optimized conditions, MCs and NOD were enriched by the MOF-5@CS material and detected by ultra-performance liquid chromatography-tandem mass spectrometry. The limit of detection of the new method for MCs and NOD were in the range of 0.0018-0.077 ng/mL. The value of relative standard deviation for repeatability were 2.69-6.30%, and the recovery of the analytes ranged from 84.36% to 118.51%. Compared with other reported method for MCs and NOD detection in complex matrices, better adsorption performance for MCs and NOD were obtained by our new method, and the sensitivity of MCs-RR and NOD were improved nearly 20 times and 30 times, respectively.


Assuntos
Quitosana/química , Cromatografia Líquida de Alta Pressão/métodos , Estruturas Metalorgânicas/química , Microcistinas/análise , Peptídeos Cíclicos/análise , Espectrometria de Massas em Tandem/métodos , Adsorção , Microcistinas/química , Peptídeos Cíclicos/química , Padrões de Referência , Reprodutibilidade dos Testes , Extração em Fase Sólida , Espectroscopia de Infravermelho com Transformada de Fourier , Poluentes Químicos da Água/análise
5.
PLoS One ; 15(4): e0232010, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32324839

RESUMO

Rheumatoid arthritis (RA), caused by the abnormal recognition of human joint cells by autoimmune antibodies, remains the world's most prevalent autoimmune disease, with over five million people affected and as much as 4% of the population at risk of RA. To prevent rapid disease development, hormonal and anti-inflammatory therapies require fast and reliable RA diagnosis. However, difficulty in detecting early specific biomarkers for RA means that it is unclear when treatment needs to begin. Here, we combined synthesis of citrullinated peptide epitopes with molecular diagnostics to verify a new specific biomarker for early RA diagnosis and flare prediction. A fibrinogen-derived 21-amino-acid-long citrullinated peptide showed high reactivity toward autoantibodies in RA samples. Additionally, the level of antibodies to this epitope was elevated prior to flares. In contrast, other citrullinated protein variants had lower reactivity and poorer sensitivity to disease activity. In conclusion, fibrinogen-derived epitope E2 subjected to citrullination facilitated a reliable RA diagnosis with a strong correlation to disease activity. This is of a high value for the diagnosis and management of RA patients who respond poorly to treatment.


Assuntos
Artrite Reumatoide/diagnóstico , Autoanticorpos/metabolismo , Epitopos/imunologia , Peptídeos Cíclicos/imunologia , Adolescente , Adulto , Artrite Reumatoide/imunologia , Biomarcadores/metabolismo , Estudos de Casos e Controles , Citrulina/metabolismo , Diagnóstico Precoce , Epitopos/química , Feminino , Fibrinogênio/química , Humanos , Masculino , Peptídeos Cíclicos/química , Índice de Gravidade de Doença , Exacerbação dos Sintomas , Adulto Jovem
6.
Nat Commun ; 11(1): 1575, 2020 03 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.


Assuntos
Biocatálise , Cisteína Endopeptidases/metabolismo , Inibidores da Tripsina/metabolismo , Sequência de Aminoácidos , Ciclização , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Peptídeos Cíclicos/química , Peptídeos Cíclicos/metabolismo , Proteínas de Plantas/metabolismo , Engenharia de Proteínas , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
7.
Yakugaku Zasshi ; 140(3): 329-344, 2020.
Artigo em Japonês | MEDLINE | ID: mdl-32115550

RESUMO

I have engaged in medicinal chemical studies based on the theoretical design of bioactive compounds. First, I present a three-dimensional structural diversity-oriented conformational restriction strategy for developing bioactive compounds based on the characteristic steric and stereoelectronic features of cyclopropane. Using this strategy, various biologically active small molecule compounds, such as receptor agonists/antagonists and enzyme inhibitors, were effectively developed. The strategy was also applied to develop versatile peptidomimetics and membrane-permeable cyclic peptides. Next, studies on Ca2+-mobilizing second messengers, cyclic ADP-ribose (cADPR) and myo-inositol trisphosphates (IP3), are described. In these studies, stable equivalents of cADPR were developed, since biological studies of cADPR have been limited due to its instability. Various potent IP3 receptor ligands, which were designed using the d-glucose structure as a bioisostere of the myo-inositol moiety of IP3, have been identified. Organic chemistry studies have also been extensively performed, because excellent organic chemistry is essential for promoting high-level medicinal chemical studies. For examples, new methods for the synthesis of chiral cyclopropanes, new radical reactions with silicon tethers, and kinetic anomeric effect-dependent stereoselective glycosidations have been developed.


Assuntos
ADP-Ribose Cíclica/química , Ciclopropanos/química , Desenho de Fármacos , Desenvolvimento de Medicamentos , Inositol 1,4,5-Trifosfato/química , Cálcio/metabolismo , Ciclopropanos/síntese química , Inibidores Enzimáticos , Glucose/química , Ligantes , Conformação Molecular , Fenômenos de Química Orgânica , Peptídeos Cíclicos/química , Peptidomiméticos , Estereoisomerismo
8.
Toxicon ; 175: 44-48, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32056695

RESUMO

A highly sensitive and broadly specific competitive indirect enzyme-linked immunosorbent assay (ciELISA) method was developed for the simultaneous detection of nine microcystins (MCs) and nodularin (NOD) using MC-LR-keyhole limpet hemocyanin (KLH) for New Zealand white rabbit immunization to produce antibodies. The MC-LR-bovine serum albumin (BSA) and NOD-BSA coating antigens were compared and heterogeneous coating strategy was found to significantly improve the sensitivity of detection, as evident from the appropriate structure. Comparison of the half-maximum inhibitory concentration (IC50) with MC-LR and MC-LR-BSA coating techniques (0.29 ng/mL) revealed the superior performance of 0.054 ng/mL for NOD-BSA coating. NOD-BSA was selected as the coating antigen, because it showed ultrahigh sensitivity for the detection of MC-LR with a limit of detection (LOD) of 0.0016 ng/mL, which was below the maximum residue level (MRL) of 1 ng/mL. In addition, high reproducibility, good stability, and acceptable spiked sample detection, as validated by liquid chromatography tandem mass spectrometry (LC-MS/MS), indicated the possible application of this method for the analysis of MCs and NOD in water sample.


Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Microcistinas/química , Peptídeos Cíclicos/química , Animais , Limite de Detecção , Coelhos , Sensibilidade e Especificidade
9.
J Med Chem ; 63(4): 1576-1596, 2020 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-32003991

RESUMO

Human cathepsin D (CatD), a pepsin-family aspartic protease, plays an important role in tumor progression and metastasis. Here, we report the development of biomimetic inhibitors of CatD as novel tools for regulation of this therapeutic target. We designed a macrocyclic scaffold to mimic the spatial conformation of the minimal pseudo-dipeptide binding motif of pepstatin A, a microbial oligopeptide inhibitor, in the CatD active site. A library of more than 30 macrocyclic peptidomimetic inhibitors was employed for scaffold optimization, mapping of subsite interactions, and profiling of inhibitor selectivity. Furthermore, we solved high-resolution crystal structures of three macrocyclic inhibitors with low nanomolar or subnanomolar potency in complex with CatD and determined their binding mode using quantum chemical calculations. The study provides a new structural template and functional profile that can be exploited for design of potential chemotherapeutics that specifically inhibit CatD and related aspartic proteases.


Assuntos
Catepsina D/antagonistas & inibidores , Catepsina D/metabolismo , Peptídeos Cíclicos/química , Peptídeos Cíclicos/metabolismo , Inibidores de Proteases/química , Inibidores de Proteases/metabolismo , Sítios de Ligação , Materiais Biomiméticos/síntese química , Materiais Biomiméticos/química , Materiais Biomiméticos/metabolismo , Materiais Biomiméticos/toxicidade , Células CACO-2 , Catepsina D/química , Ensaios Enzimáticos , Humanos , Cinética , Estrutura Molecular , Pepstatinas/química , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/toxicidade , Inibidores de Proteases/síntese química , Inibidores de Proteases/toxicidade , Ligação Proteica , Relação Estrutura-Atividade
10.
J Chromatogr A ; 1618: 460902, 2020 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-32067759

RESUMO

Complexity and diversity of natural compounds make it a challenge for globally profiling constituents in multiple species plants, especially for minor new compounds. Rubiaceae-type cyclopeptides (RAs) are one kind of active constituents and characteristic components in Rubia plants, particularly Rubia cordifolia (RC), which is one kind of traditional Chinese medicines. In this study, a new multiple reaction monitoring strategy (PPCP-MRM) based on predicted precursor ions and characteristic product ions was developed to globally profile RAs in RC and its two main adulterants, including Rubia alata (also named Jinjiancao in Chinese) (RAJ) and Rubia podantha (RP). Moreover, characteristic components of these species have been found by targeted relative quantitative analysis of RAs by LC-MS. In total, 39 RAs have been structurally annotated based on fragment ions in MS2 data, including 19 new compounds. In addition, 7 RAs as the chemical markers were found to distinguish these three Rubia species. The results indicated that this PPCP-MRM integrated strategy is a powerful tool for comprehensive analysis of RAs and screening chemical markers for Rubia species discrimination, which would be useful for distinguishing Rubia adulterants. Furthermore, this developed strategy could also be a useful tool for analysis of other cyclopeptides.


Assuntos
Peptídeos Cíclicos/química , Rubia/química , Biomarcadores/química , Cromatografia Líquida , Íons , Espectrometria de Massas
11.
Nature ; 578(7796): 582-587, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32051588

RESUMO

Addressing the ongoing antibiotic crisis requires the discovery of compounds with novel mechanisms of action that are capable of treating drug-resistant infections1. Many antibiotics are sourced from specialized metabolites produced by bacteria, particularly those of the Actinomycetes family2. Although actinomycete extracts have traditionally been screened using activity-based platforms, this approach has become unfavourable owing to the frequent rediscovery of known compounds. Genome sequencing of actinomycetes reveals an untapped reservoir of biosynthetic gene clusters, but prioritization is required to predict which gene clusters may yield promising new chemical matter2. Here we make use of the phylogeny of biosynthetic genes along with the lack of known resistance determinants to predict divergent members of the glycopeptide family of antibiotics that are likely to possess new biological activities. Using these predictions, we uncovered two members of a new functional class of glycopeptide antibiotics-the known glycopeptide antibiotic complestatin and a newly discovered compound we call corbomycin-that have a novel mode of action. We show that by binding to peptidoglycan, complestatin and corbomycin block the action of autolysins-essential peptidoglycan hydrolases that are required for remodelling of the cell wall during growth. Corbomycin and complestatin have low levels of resistance development and are effective in reducing bacterial burden in a mouse model of skin MRSA infection.


Assuntos
Antibacterianos , Descoberta de Drogas , Peptídeos Cíclicos , Peptidoglicano/efeitos dos fármacos , Peptidoglicano/metabolismo , Actinobacteria/química , Actinobacteria/genética , Actinobacteria/metabolismo , Animais , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Vias Biossintéticas/genética , Parede Celular/metabolismo , Clorofenóis/química , Clorofenóis/metabolismo , Clorofenóis/farmacologia , Modelos Animais de Doenças , Resistência Microbiana a Medicamentos/efeitos dos fármacos , Resistência Microbiana a Medicamentos/genética , Feminino , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Camundongos , Testes de Sensibilidade Microbiana , Família Multigênica , N-Acetil-Muramil-L-Alanina Amidase/antagonistas & inibidores , Peptídeos Cíclicos/química , Peptídeos Cíclicos/metabolismo , Peptídeos Cíclicos/farmacologia , Filogenia , Pele/microbiologia , Infecções Estafilocócicas/microbiologia
12.
Chemistry ; 26(23): 5231-5244, 2020 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-32027758

RESUMO

It has been hypothesised that drugs in the chemical space "beyond the rule of 5" (bRo5) must behave as molecular chameleons to combine otherwise conflicting properties, including aqueous solubility, cell permeability and target binding. Evidence for this has, however, been limited to the cyclic peptide cyclosporine A. Herein, we show that the non-peptidic and macrocyclic drugs roxithromycin, telithromycin and spiramycin behave as molecular chameleons, with rifampicin showing a less pronounced behaviour. In particular roxithromycin, telithromycin and spiramycin display a marked, yet limited flexibility and populate significantly less polar and more compact conformational ensembles in an apolar than in a polar environment. In addition to balancing of membrane permeability and aqueous solubility, this flexibility also allows binding to targets that vary in structure between species. The drugs' passive cell permeability correlates to their 3D polar surface area and corroborate two theoretical models for permeability, developed for cyclic peptides. We conclude that molecular chameleonicity should be incorporated in the design of orally administered drugs in the bRo5 space.


Assuntos
Lagartos/metabolismo , Peptídeos Cíclicos/química , Administração Oral , Animais , Permeabilidade da Membrana Celular , Conformação Molecular , Permeabilidade , Solubilidade
13.
Biochim Biophys Acta Proteins Proteom ; 1868(5): 140378, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32032759

RESUMO

Amyloidogenic disorders are currently rising as a global health issue, prompting more and more studies dedicated to the development of effective targeted therapeutics. The innate affinity of these amyloidogenic proteins towards the biomembranes adds further complexities to the systems. Our previous studies have shown that biologically active peptides can effectively target amyloidogenesis serving as an efficient therapeutic alternative in several amyloidogenic disorders. The structural uniqueness of the PWWP motif in the de novo designed heptapeptide, KR7 (KPWWPRR-NH2) was demonstrated to target insulin fiber elongation specifically. By working on insulin, an important model system in amyloidogenic studies, we gained several mechanistic insights into the inhibitory actions at the protein-peptide interface. Here, we report a second-generation non-toxic and serum stable cyclic peptide, based primarily on the PWWP motif that resulted in complete inhibition of insulin fibrillation both in the presence and absence of the model membranes. Using both low- and high-resolution spectroscopic techniques, we could delineate the specific mechanism of inhibition, at atomistic resolution. Our studies put forward an effective therapeutic intervention that redirects the default aggregation kinetics towards off-pathway fibrillation. Based on the promising results, this novel cyclic peptide can be considered an excellent lead to design pharmaceutical molecules against amyloidogenesis.


Assuntos
Amiloide/química , Insulina/química , Peptídeos Cíclicos/farmacologia , Peptídeos Cíclicos/química , Multimerização Proteica/efeitos dos fármacos
14.
Chem Commun (Camb) ; 56(12): 1788-1791, 2020 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-31960841

RESUMO

In this study, αvß3 integrin in U87 tumor cells was imaged with a 64Cu-peptidic probe, in which the linear peptide GHRGDHG is used as a pre-ligand, while 64Cu bears three functional roles that include generation of the PET signal, coordination with two GH moieties of the pre-ligand, and cyclizing the linear pre-ligand into an active cyclic-RGD form (termed as 64Cu-Cyclo-RGD) for αvß3 integrin.


Assuntos
Radioisótopos de Cobre/química , Corantes Fluorescentes/química , Integrina beta3/análise , Peptídeos Cíclicos/química , Tomografia por Emissão de Pósitrons , Linhagem Celular Tumoral , Humanos , Imagem Óptica
15.
Science ; 367(6476): 458-463, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31896661

RESUMO

Molecular shape defines function in both biological and material settings, and chemists have developed an ever-increasing vernacular to describe these shapes. Noncanonical atropisomers-shape-defined molecules that are formally topologically trivial but are interconvertible only by complex, nonphysical multibond torsions-form a unique subset of atropisomers that differ from both canonical atropisomers (e.g., binaphthyls) and topoisomers (i.e., molecules that have identical connectivity but nonidentical molecular graphs). Small molecules, in contrast to biomacromolecules, are not expected to exhibit such ambiguous shapes. Using total synthesis, we found that the peptidic alkaloid tryptorubin A can be one of two noncanonical atropisomers. We then devised a synthetic strategy that drives the atropospecific synthesis of a noncanonical atrop-defined small molecule.


Assuntos
Produtos Biológicos/metabolismo , Peptídeos Cíclicos/biossíntese , Sequência de Aminoácidos , Produtos Biológicos/química , Peptídeos Cíclicos/química , Peptídeos Cíclicos/genética , Estereoisomerismo , Streptomyces/genética , Streptomyces/metabolismo , Xanthomonas/genética , Xanthomonas/metabolismo
16.
Chemistry ; 26(12): 2602-2605, 2020 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-31943410

RESUMO

The effective delivery of cytotoxic agents to tumor cells is a key challenge in anticancer therapy. Multivalent integrinspecific ligands are considered a promising tool to increase the binding affinity, selectivity, and internalization efficiency of small-molecule drug conjugates. Herein, we report the synthesis and biological evaluation of a multimeric conjugate containing the high-affinity integrin αv ß3 binding ligand RAFT-c(RGDfK)4 , a lysosomally cleavable Val-Cit linker, and cryptophycin-55 glycinate, a potent inhibitor of tubulin polymerization. In vitro cytotoxicity assays verified that the multimeric RGD-cryptophycin conjugate displays improved potency compared to the monomeric analogue in integrin αv ß3 overexpressing tumor cell lines, while significantly reduced activity was observed in the integrin-negative cell line.


Assuntos
Depsipeptídeos/química , Portadores de Fármacos/química , Peptídeos Cíclicos/química , Linhagem Celular Tumoral , Liberação Controlada de Fármacos , Humanos , Integrina alfaVbeta3/química , Terapia de Alvo Molecular/métodos
17.
Microb Cell Fact ; 19(1): 5, 2020 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-31918711

RESUMO

BACKGROUND: Heterologous expression of secondary metabolite gene clusters is used to achieve increased production of desired compounds, activate cryptic gene clusters, manipulate clusters from genetically unamenable strains, obtain natural products from uncultivable species, create new unnatural pathways, etc. Several Streptomyces species are genetically engineered for use as hosts for heterologous expression of gene clusters. S. lividans TK24 is one of the most studied and genetically tractable actinobacteria, which remain untapped. It was therefore important to generate S. lividans chassis strains with clean metabolic backgrounds. RESULTS: In this study, we generated a set of S. lividans chassis strains by deleting endogenous gene clusters and introducing additional φC31 attB loci for site-specific integration of foreign DNA. In addition to the simplified metabolic background, the engineered S. lividans strains had better growth characteristics than the parental strain in liquid production medium. The utility of the developed strains was validated by expressing four secondary metabolite gene clusters responsible for the production of different classes of natural products. Engineered strains were found to be superior to the parental strain in production of heterologous natural products. Furthermore, S. lividans-based strains were better producers of amino acid-based natural products than other tested common hosts. Expression of a Streptomyces albus subsp. chlorinus NRRL B-24108 genomic library in the modified S. lividans ΔYA9 and S. albus Del14 strains resulted in the production of 7 potentially new compounds, only one of which was produced in both strains. CONCLUSION: The constructed S. lividans-based strains are a great complement to the panel of heterologous hosts for actinobacterial secondary metabolite gene expression. The expansion of the number of such engineered strains will contribute to an increased success rate in isolation of new natural products originating from the expression of genomic and metagenomic libraries, thus raising the chance to obtain novel biologically active compounds.


Assuntos
Antibacterianos/biossíntese , Produtos Biológicos , Metabolismo Secundário/genética , Streptomyces lividans/genética , Actinobacteria/genética , Actinobacteria/metabolismo , Antibacterianos/química , Bacteriocinas/biossíntese , Bacteriocinas/química , Produtos Biológicos/química , Produtos Biológicos/metabolismo , Clonagem Molecular , Engenharia Genética/métodos , Família Multigênica , Peptídeos Cíclicos/biossíntese , Peptídeos Cíclicos/química , Streptomyces lividans/metabolismo , Tunicamicina/biossíntese , Tunicamicina/química
18.
Phys Chem Chem Phys ; 22(6): 3570-3583, 2020 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-31995079

RESUMO

Despite being recognized as a therapeutic target in the processes of cancer cell proliferation and metastasis for over 50 years, the interaction of the urokinase plasminogen activator uPA with its receptor uPAR still needs an improved understanding. High resolution crystallographic data (PDB ) of the uPA-uPAR binding geometry was used to perform quantum biochemistry computations within the density functional theory (DFT) framework. A divide to conquer methodology considering a mixed homogeneous/inhomogeneous dielectric model and explicitly taking water molecules into account was employed to obtain a large set of uPA-uPAR residue-residue interaction energies. In order of importance, not only were Phe25 > Tyr24 > Trp30 > Ile28 shown to be the most relevant uPA residues binding it to uPAR, but the residues Lys98 > His87 > Gln40 > Asn22 > Lys23 > Val20 also had significant interaction energies, which helps to explain published experimental mutational data. Furthermore, the results obtained with the uPA-uPAR in/homogeneous dielectric function show that a high dielectric constant value ε = 40 is adequate to take into account the electrostatic environment at the interface between the proteins, while using a smaller value of ε (<10) leads to an overestimation of the uPA-uPAR binding energy. Hot spots of the uPA-uPAR binding domain were identified and a quantum biochemistry description of the uPAR blockers uPA21-30 and cyclo21,29uPA21-29[(S21C;H29C)] was performed, demonstrating that cyclization improves the stability of mimetic peptides without compromising their binding energies to uPAR.


Assuntos
Receptores de Ativador de Plasminogênio Tipo Uroquinase/química , Ativador de Plasminogênio Tipo Uroquinase/química , Sequência de Aminoácidos , Aminoácidos/química , Teoria da Densidade Funcional , Peptídeos Cíclicos/química , Ligação Proteica , Conformação Proteica , Eletricidade Estática , Relação Estrutura-Atividade , Termodinâmica
20.
Chemistry ; 26(26): 5846-5858, 2020 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-31999874

RESUMO

Cyclic peptides with disc-shaped structures have emerged as potent building blocks for the preparation of new biomaterials in fields ranging from biological to material science. In this work, we analyze in depth the self-assembling properties of a new type of cyclic peptides based on the alternation of α-residues and cyclic δ-amino acids (α,δ-CPs). To examine the preferred stacking properties adopted by cyclic peptides bearing this type of amino acids, we carried out a synergistic in vitro/in silico approximation by using simple dimeric models and then extended to nanotubes. Although these new cyclic peptides (α,δ-CPs) can interact either in a parallel or antiparallel fashion, our results confirm that although the parallel ß-sheet is more stable, it can be switched to the antiparallel stacking by choosing residues that can establish favorable cross-strand interactions. Moreover, the subsequent comparison by using the same methodology but applied to α,γ-CPs models, up to the moment assumed as antiparallel-like d,l-α-CPs, led to unforeseen conclusions that put into question preliminary conjectures about these systems. Surprisingly, they tend to adopt a parallel ß-sheet directed by the skeleton interactions. These results imply a change of paradigm with respect to cyclic peptide designs that should be considered for dimers and nanotubes.


Assuntos
Aminoácidos Cíclicos/química , Peptídeos Cíclicos/química , Proteínas/química , Simulação por Computador , Ligação de Hidrogênio , Conformação Proteica em Folha beta
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