RESUMO
Considering the intrinsic toxicities of transition metals, their incorporation into drug therapies must operate at minimal amounts while ensuring adequate catalytic activity within complex biological systems. As a way to address this issue, this study investigates the design of synthetic prodrugs that are not only tuned to be harmless, but can be robustly transformed in vivo to reach therapeutically relevant levels. To accomplish this, retrosynthetic prodrug design highlights the potential of naphthylcombretastatin-based prodrugs, which form highly active cytostatic agents via sequential ring-closing metathesis and aromatization. Structural adjustments will also be done to improve aspects related to catalytic reactivity, intrinsic bioactivity, and hydrolytic stability. The developed prodrug therapy is found to possess excellent anticancer activities in cell-based assays. Furthermore, in vivo activation by intravenously administered glycosylated artificial metalloenzymes can also induce significant reduction of implanted tumor growth in mice.
Assuntos
Antineoplásicos/química , Biocatálise , Neoplasias/tratamento farmacológico , Pró-Fármacos/química , Animais , Antineoplásicos/farmacologia , Fenômenos Biológicos , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos , Desenho de Fármacos , Desenvolvimento de Medicamentos , Descoberta de Drogas , Feminino , Humanos , Recém-Nascido , Doenças do Recém-Nascido , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Simulação de Acoplamento Molecular , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
This study presents the early framework of selective cell tagging (SeCT) therapy, which is the concept of preferentially labeling specific cells in vivo with chemical moieties that can elicit a therapeutic response. Using glycosylated artificial metalloenzyme (GArM)-based protein labeling, this study reports two separate functional strategies. In one approach, early tumor onset can be suppressed by tagging cancer cells in living mice with an integrin-blocking cyclic-Arg-Gly-Asp (cRGD) moiety, thereby disrupting cell adhesion onto the extracellular matrix. In another approach, tumor growth in mice can be reduced by tagging with a cytotoxic doxorubicin moiety. Subsequent cell death occurs following internalization and drug release. Overall, experiments have shown that mouse populations receiving the mixture of SeCT labeling reagents exhibited a significant delay/reduction in tumor onset and growth compared with controls. Highlighting its adaptability, this work represents a foundational step for further development of SeCT therapy and its potential therapeutic applications.
RESUMO
An emerging approach in the field of targeted drug delivery is the establishment of abiotic metal-triggered prodrug mechanisms that can control the release of bioactive drugs. Currently, the design of prodrugs that use abiotic metals as a trigger relies heavily on uncaging strategies. Here, we introduce a strategy based on the gold-catalyzed activation of a phenanthridinium-based prodrug via hydroamination under physiological conditions. To make the prodrug strategy biocompatible, a gold artificial metalloenzyme (ArM) based on human serum albumin, rather than the free gold metal complex, was used as a trigger for prodrug activation. The albumin-based gold ArM protected the catalytic activity of the bound gold metal even in the presence of up to 1â mM glutathione in vitro. The drug synthesized via the gold ArM exerted a therapeutic effect in cell-based assays, highlighting the potential usefulness of the gold ArM in anticancer applications.
Assuntos
Ouro/química , Fenantridinas/química , Pró-Fármacos/química , Células A549 , Aminação , Catálise , Sobrevivência Celular/efeitos dos fármacos , Ciclização , Glutationa/química , Humanos , Metaloproteínas/química , Metaloproteínas/metabolismo , Pró-Fármacos/síntese química , Pró-Fármacos/farmacologia , Albumina Sérica/químicaRESUMO
In order to harness the functionality of metals, nature has evolved over billions of years to utilize metalloproteins as key components in numerous cellular processes. Despite this, transition metals such as ruthenium, palladium, iridium, and gold are largely absent from naturally occurring metalloproteins, likely due to their scarcity as precious metals. To mimic the evolutionary process of nature, the field of artificial metalloenzymes (ArMs) was born as a way to benefit from the unique chemoselectivity and orthogonality of transition metals in a biological setting. In its current state, numerous examples have successfully incorporated transition metals into a variety of protein scaffolds. Using these ArMs, many examples of new-to-nature reactions have been carried out, some of which have shown substantial biocompatibility. Given the rapid rate at which this field is growing, this review aims to highlight some important studies that have begun to take the next step within this field; namely the development of ArM-centered drug therapies or biotechnological tools.
Assuntos
Materiais Biomiméticos/química , Coenzimas/química , Metaloproteínas/química , Metais/química , Biocatálise , Técnicas Biossensoriais , Modelos Moleculares , Conformação Proteica , Engenharia de Proteínas , EstereoisomerismoRESUMO
Akin to a cellular "fingerprint," the glycocalyx is a glycan-enriched cellular coating that plays a crucial role in mediating cell-to-cell interactions. To gain a better understanding of the factors that govern in vivo recognition, artificial glycoproteins were initially created to probe changes made to the accumulation and biodistribution of specific glycan assemblies through biomimicry. As a result, the organ-specific accumulation for a variety of glycoproteins decorated with simple and/or complex glycans was identified. Additionally, binding trends with regard to cancer cell selectivity were also investigated. To exploit the knowledge gained from these studies, numerous groups thus became engaged in developing targeted drug methodologies based on the use of artificial glycoproteins. This has either been done through adopting the glycoprotein scaffold as a drug carrier, or to directly glycosylate therapeutic proteins/enzymes to localize their biological activity. The principle aim of this Review is to present the foundational research that has driven artificial glycoprotein-based targeting and subsequent adaptations with potential therapeutic applications.
Assuntos
Sistemas de Liberação de Medicamentos , Glicoproteínas , Sistemas de Liberação de Medicamentos/métodos , Glicocálix/química , Glicocálix/metabolismo , Glicoproteínas/química , Humanos , Neoplasias/tratamento farmacológico , Distribuição TecidualRESUMO
Metal-based uncaging of biomolecules has become an emerging approach for in vivo applications, which is largely due to the advantageous bioorthogonality of abiotic transition metals. Adding to the library of metal-cleavable protecting groups, this work introduces the 2-alkynylbenzamide (Ayba) moiety for the gold-triggered release of secondary amines under mild and physiological conditions. Studies were further performed to highlight some intrinsic benefits of the Ayba protecting group, which are (1) its amenable nature to derivatization for manipulating prodrug properties, and (2) its orthogonality with other commonly used transition metals like palladium and ruthenium. With a focus on highlighting its application for anticancer drug therapies, this study successfully shows that gold-triggered conversion of Ayba-protected prodrugs into bioactive anticancer drugs (i.e. doxorubicin, endoxifen) can proceed effectively in cell-based assays.
RESUMO
Enzyme biosensors are useful tools that can monitor rapid changes in metabolite levels in real-time. However, current approaches are largely constrained to metabolites within a limited chemical space. With the rising development of artificial metalloenzymes (ArM), a unique opportunity exists to design biosensors from the ground-up for metabolites that are difficult to detect using current technologies. Here we present the design and development of the ArM ethylene probe (AEP), where an albumin scaffold is used to solubilize and protect a quenched ruthenium catalyst. In the presence of the phytohormone ethylene, cross metathesis can occur to produce fluorescence. The probe can be used to detect both exogenous- and endogenous-induced changes to ethylene biosynthesis in fruits and leaves. Overall, this work represents an example of an ArM biosensor, designed specifically for the spatial and temporal detection of a biological metabolite previously not accessible using enzyme biosensors.
Assuntos
Materiais Biomiméticos/síntese química , Técnicas Biossensoriais/instrumentação , Etilenos/análise , Metaloproteínas/metabolismo , Reguladores de Crescimento de Plantas/análise , Actinidia/metabolismo , Arabidopsis/metabolismo , Catálise , Técnicas de Química Sintética/métodos , Enzimas/síntese química , Enzimas/metabolismo , Etilenos/metabolismo , Fluorescência , Frutas/metabolismo , Gases/análise , Gases/metabolismo , Metaloproteínas/síntese química , Reguladores de Crescimento de Plantas/metabolismo , Folhas de Planta/química , Rutênio/química , Albumina Sérica Humana/síntese química , Albumina Sérica Humana/metabolismoRESUMO
Aminoglycosides are a group of broad-spectrum antibiotics that have been used in the clinic for almost a century. The rapid spread of bacterial genes coding for aminoglycoside-modifying enzymes has, however, dramatically decreased the utility of aminoglycosides. We have previously reported several aminoglycoside potentiators that work by inhibiting aminoglycoside N-6'-acetyltransferase, one of the most common determinants of aminoglycoside resistance. Among these, prodrugs that combine the structure of an aminoglycoside with that of pantothenate into one molecule are especially promising. We report here a series of cellular studies to investigate the activity and mechanism of action of these prodrugs further. Our results reveal a new aminoglycoside resistance inhibitor, as well as the possibility that these prodrugs are transformed into more than one inhibitor in bacteria. We also report that the onset of the potentiators is rapid. Their low cell cytotoxicity, good stability, and potentiation of various aminoglycosides, against both Gram-positive and Gram-negative bacteria, make them interesting compounds for the development of new drugs.
Assuntos
Acetiltransferases/efeitos dos fármacos , Antibacterianos , Farmacorresistência Bacteriana/efeitos dos fármacos , Enterococcus faecium/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Pró-Fármacos , Aminoglicosídeos/metabolismo , Antibacterianos/química , Antibacterianos/farmacologia , Células HeLa , Humanos , Pró-Fármacos/química , Pró-Fármacos/farmacologiaRESUMO
This work represents the first broad study of testing diverse heterogenous glycoconjugates (7 different glycoalbumins) for their differential in vivo binding (11 different cancer cell types) in both cell- and animal-based studies. As a result, various changes in biodistribution, excretion, and even tumor adhesion were observed.
Assuntos
Moléculas de Adesão Celular/farmacocinética , Glicoconjugados/farmacocinética , Albumina Sérica/farmacocinética , Animais , Moléculas de Adesão Celular/química , Linhagem Celular Tumoral , Produtos Finais de Glicação Avançada , Glicoconjugados/química , Humanos , Camundongos , Especificidade de Órgãos , Albumina Sérica/química , Distribuição Tecidual , Albumina Sérica GlicadaRESUMO
In previously reported work, AuIII complexes coordinated with 2-benzoylpyridine ligand, BPy-Au, were prebound to a protein and used to discover a novel protein-directed labeling approach with propargyl ester functional groups. In this work, further examination discovered that gold catalysts devoid of the 2-benzoylpyridine ligand (e.g., NaAuCl4) had significantly reduced levels of protein labeling. Mechanistic investigations then revealed that BPy-Au and propargyl esters undergo a rare example of C(sp2 )-C(sp) aryl-alkynyl cross-coupling, likely through spontaneous reductive elimination. Overall, these observations appear to suggest that BPy-Au-mediated, propargyl ester-based protein labeling acts via an activated ester intermediate, which contributes to our understanding of this process and will aid the expansion/optimization of gold-catalyst usage in future bioconjugation applications, especially in vivo.
Assuntos
Ouro/química , Pargilina/química , Piridinas/química , Catálise , Ésteres , Ligantes , Coloração e RotulagemRESUMO
Rapidly growing cancer cells have increased levels of intracellular polyamines compared to normal, healthy tissues. Based on the selective reactivity of glycine propargyl esters, probes were synthesized that show evidence for selective polyamine reactivity, which was then applied for selective cancer cell imaging studies.
RESUMO
Metal complex catalysis within biological systems is largely limited to cell and bacterial systems. In this work, a glycoalbumin-AuIII complex was designed and developed that enables organ-specific, localized propargyl ester amidation with nearby proteins within live mice. The targeted reactivity can be imaged through the use of Cy7.5- and TAMRA-linked propargyl ester based fluorescent probes. This targeting system could enable the exploitation of other metal catalysis strategies for biomedical and clinical applications.
Assuntos
Complexos de Coordenação/química , Corantes Fluorescentes/química , Ouro/química , Albumina Sérica/química , Animais , Catálise , Complexos de Coordenação/farmacocinética , Corantes Fluorescentes/farmacocinética , Produtos Finais de Glicação Avançada , Ouro/farmacocinética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Imagem Óptica/métodos , Albumina Sérica/farmacocinética , Distribuição Tecidual , Albumina Sérica GlicadaRESUMO
Alkyl esters, such as propargyl esters, typically lack the electron-withdrawing inductive effects needed to participate in nucleophilic acyl substitution reactions. Herein, we report an unusual observation in which glycine propargyl ester derivatives displayed selective, base-independent reactivity towards linear alkylamines under mild, metal-free conditions. Through global reaction route mapping (GRRM) modeling calculations, it is predicted that these observations may be governed by factors related to hydrogen-bonding and intermolecular interactions, rather than electron-withdrawing inductive effects. Based on this concept of propargyl-assisted selective amidation, a direct application was made to develop a novel site-specific C-terminal glycine peptide bioconjugation technique as a proof-of-concept, which relies upon the selective reactivity of glycine propargyl esters over that of aspartate and glutamate side-chain-linked propargyl esters.
Assuntos
Amidas/química , Glicina/química , Pargilina/química , Peptídeos/química , Elétrons , Ésteres , Glutamatos , Ligação de HidrogênioRESUMO
While many examples have been reported that glycoclusters interact with target lectins more strongly than single molecules of glycans, through multivalency effects, literature examples to support lectin interactions/modulations on cell surface and in live animals is quite rare. Our N-glycoclusters, which were efficiently prepared by immobilizing 16 molecules of the asparagine-linked glycans (N-glycans) onto a lysine-based dendron template through histidine-mediated Huisgen cycloaddition, were shown to efficiently detect platelet endothelial cell adhesion molecule (PECAM) on human umbilical vein endothelial cells (HUVEC) as a α(2-6)-sialylated oligosaccharides recognizing lectin. Furthermore, the identity of the N-glycans on our N-glycoclusters allowed control over organ-selective accumulation and serum clearance properties when intravenously injected into mice.
Assuntos
Lectinas/metabolismo , Glicoproteínas de Membrana/metabolismo , Polissacarídeos/metabolismo , Animais , Asparagina/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Dendrímeros/química , Dendrímeros/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Lectinas/química , Glicoproteínas de Membrana/química , Camundongos , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Polissacarídeos/químicaRESUMO
The coenzyme A (CoA) biosynthetic enzymes have been used to produce various CoA analogues, including mechanistic probes of CoA-dependent enzymes such as those involved in fatty acid biosynthesis. These enzymes are also important for the activation of the pantothenamide class of antibacterial agents, and of a recently reported family of antibiotic resistance inhibitors. Herein we report a study on the selectivity of pantothenate kinase, the first and rate limiting step of CoA biosynthesis. A robust synthetic route was developed to allow rapid access to a small library of pantothenate analogs diversified at the ß-alanine moiety, the carboxylate or the geminal dimethyl group. All derivatives were tested as substrates of Escherichia coli pantothenate kinase (EcPanK). Four derivatives, all N-aromatic pantothenamides, proved to be equivalent to the benchmark N-pentylpantothenamide (N5-pan) as substrates of EcPanK, while two others, also with N-aromatic groups, were some of the best substrates reported for this enzyme. This collection of data provides insight for the future design of PanK substrates in the production of useful CoA analogues.
Assuntos
Escherichia coli/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/química , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Domínio Catalítico , Coenzima A/metabolismo , Modelos Moleculares , Ácido Pantotênico/análogos & derivados , Ácido Pantotênico/metabolismo , Estrutura Terciária de Proteína , Relação Estrutura-AtividadeRESUMO
The most common mechanism of resistance to aminoglycoside antibiotics entails bacterial expression of drug-metabolizing enzymes, such as the clinically widespread aminoglycoside N-6'-acetyltransferase (AAC(6')). Aminoglycoside-CoA bisubstrates are highly potent AAC(6') inhibitors; however, their inability to penetrate cells precludes in vivo studies. Some truncated bisubstrates are known to cross cell membranes, yet their activities against AAC(6') are in the micromolar range at best. We report here the synthesis and biological activity of aminoglycoside-pantetheine derivatives that, although devoid of AAC(6') inhibitory activity, can potentiate the antibacterial activity of kanamycin A against an aminoglycoside-resistant strain of Enterococcus faecium. Biological studies demonstrate that these molecules are potentially extended to their corresponding full-length bisubstrates by enzymes of the coenzyme A biosynthetic pathway. This work provides a proof-of-concept for the utility of prodrug compounds activated by enzymes of the coenzyme A biosynthetic pathway, to resensitize resistant strains of bacteria to aminoglycoside antibiotics.
Assuntos
Aminoglicosídeos/farmacologia , Antibacterianos/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Enterococcus faecium/efeitos dos fármacos , Canamicina/farmacologia , Pró-Fármacos/metabolismo , Pró-Fármacos/farmacologia , Acetiltransferases/antagonistas & inibidores , Acetiltransferases/metabolismo , Aminoglicosídeos/síntese química , Aminoglicosídeos/química , Antibacterianos/síntese química , Antibacterianos/química , Coenzima A/biossíntese , Coenzima A/metabolismo , Relação Dose-Resposta a Droga , Enterococcus faecium/citologia , Enterococcus faecium/enzimologia , Canamicina/síntese química , Canamicina/química , Panteteína/síntese química , Panteteína/química , Panteteína/farmacologia , Relação Estrutura-AtividadeRESUMO
Aminoglycosides occupy a special niche amongst antibiotics in part because of their broad spectrum of action. Bacterial resistance is however menacing to render these drugs obsolete. A significant amount of work has been devoted to understand and overcome aminoglycoside resistance. This mini-review will discuss aminoglycoside-modifying enzymes (AMEs), with a special emphasis on the efforts to comprehend and block resistance caused by aminoglycoside 6'-N-acetyltransferase (AAC(6')).
RESUMO
As a key precursor of coenzyme A (CoA) biosynthesis, pantothenic acid has proven to be a useful backbone to elaborate probes of this biosynthetic pathway, study CoA-utilizing systems, and design molecules with antimicrobial activity. The increasing prevalence of bacterial strains resistant to one or more antibiotics has prompted a renewed interest for molecules with a novel mode of antibacterial action such as N-substituted pantothenamides. Although numerous derivatives have been reported, most are varied at the terminal N-substituent, and fewer at the ß-alanine moiety. Modifications at the pantoyl portion are limited to the addition of an ω-methyl group. We report a synthetic route to N-substituted pantothenamides with various alkyl substituents replacing the geminal dimethyl groups. Our methodology is also applicable to the synthesis of pantothenic acid, pantetheine and CoA derivatives. Here a small library of new N-substituted pantothenamides was synthesized. Most of these compounds display antibacterial activity against sensitive and resistant Staphylococcus aureus. Interestingly, replacement of the ProR methyl with an allyl group yielded a new N-substituted pantothenamide which is amongst the most potent reported so far.
Assuntos
Antibacterianos/síntese química , Ácido Pantotênico/síntese química , Ácido Pantotênico/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Amidas , Antibacterianos/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Métodos , Ácido Pantotênico/química , Bibliotecas de Moléculas Pequenas/síntese química , Relação Estrutura-AtividadeRESUMO
Aminoglycoside-coenzyme A conjugates are challenging synthetic targets because of the wealth of functional groups and high polarity of the starting materials. We previously reported a one-pot synthesis of amide-linked aminoglycoside-CoA bisubstrates. These molecules are nanomolar inhibitors of aminoglycoside N-6'-acetyltransferase Ii (AAC(6')-Ii), an important enzyme involved in bacterial resistance to aminoglycoside antibiotics. We report here the synthesis and biological activity of five new aminoglycoside-CoA bisubstrates containing sulfonamide, sulfoxide, or sulfone groups. Interestingly, the sulfonamide-linked bisubstrate, which was expected to best mimic the tetrahedral intermediate, does not show improved inhibition when compared with amide-linked bisubstrates. On the other hand, most of the sulfone- and sulfoxide-containing bisubstrates prepared are nanomolar inhibitors of AAC(6')-Ii.