RESUMO
Idiopathic pulmonary fibrosis (IPF) is a lethal disease with an average life expectancy of 3 to 5 years. IPF is characterized by progressive stiffening of the lung parenchyma due to excessive deposition of collagen, leading to gradual failure of gas exchange. Although two therapeutic agents have been approved from the FDA for IPF, they only slow disease progression with little impact on outcome. To develop a more effective therapy, we have exploited the fact that collagen-producing myofibroblasts express a membrane-spanning protein, fibroblast activation protein (FAP), that exhibits limited if any expression on other cell types. Because collagen-producing myofibroblasts are only found in fibrotic tissues, solid tumors, and healing wounds, FAP constitutes an excellent marker for targeted delivery of drugs to tissues undergoing pathologic fibrosis. We demonstrate here that a low-molecular weight FAP ligand can be used to deliver imaging and therapeutic agents selectively to FAP-expressing cells. Because induction of collagen synthesis is associated with phosphatidylinositol 3-kinase (PI3K) activation, we designed a FAP-targeted PI3K inhibitor that selectively targets FAP-expressing human IPF lung fibroblasts and potently inhibited collagen synthesis. Moreover, we showed that administration of the inhibitor in a mouse model of IPF inhibited PI3K activation in fibrotic lungs, suppressed production of hydroxyproline (major building block of collagen), reduced collagen deposition, and increased mouse survival. Collectively, these studies suggest that a FAP-targeted PI3K inhibitor might be promising for treating IPF.
Assuntos
Fibrose Pulmonar Idiopática , Fosfatidilinositol 3-Quinases , Animais , Fibroblastos , Fibrose Pulmonar Idiopática/tratamento farmacológico , Pulmão , Camundongos , Modelos Teóricos , Serina-Treonina Quinases TORRESUMO
Fibrotic diseases cause organ failure that lead to ~45% of all deaths in the United States. Activated macrophages stimulate fibrosis by secreting cytokines that induce fibroblasts to synthesize collagen and extracellular matrix proteins. Although suppression of macrophage-derived cytokine production can halt progression of fibrosis, therapeutic agents that prevent release of these cytokines (e.g., TLR7 agonists) have proven too toxic to administer systemically. Based on the expression of folate receptor ß solely on activated myeloid cells, we have created a folate-targeted TLR7 agonist (FA-TLR7-54) that selectively accumulates in profibrotic macrophages and suppresses fibrosis-inducing cytokine production. We demonstrate that FA-TLR7-54 reprograms M2-like fibrosis-inducing macrophages into fibrosis-suppressing macrophages, resulting in dramatic declines in profibrotic cytokine release, hydroxyproline biosynthesis, and collagen deposition, with concomitant increases in alveolar airspaces. Although nontargeted TLR7-54 is lethal at fibrosis-suppressing doses, FA-TLR7-54 halts fibrosis without evidence of toxicity. Taken together, FA-TLR7-54 is shown to constitute a novel and potent approach for treating fibrosis without causing dose-limiting systemic toxicities.
Assuntos
Bleomicina , Fibrose Pulmonar , Animais , Fibroblastos , Macrófagos , Macrófagos Alveolares , Camundongos , Camundongos Endogâmicos C57BL , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/tratamento farmacológicoRESUMO
Background: Cancer-associated fibroblasts (CAFs) comprise a major cell type in the tumor microenvironment where they support tumor growth and survival by producing extracellular matrix, secreting immunosuppressive cytokines, releasing growth factors, and facilitating metastases. Because tumors with elevated CAFs are characterized by poorer prognosis, considerable effort is focused on developing methods to quantitate, suppress and/or eliminate CAFs. We exploit the elevated expression of fibroblast activation protein (FAP) on CAFs to target imaging and therapeutic agents selectively to these fibroblasts in solid tumors. Methods: FAP-targeted optical imaging, radioimaging, and chemotherapeutic agents were synthesized by conjugating FAP ligand (FL) to either a fluorescent dye, technetium-99m, or tubulysin B hydrazide. In vitro and in vivo studies were performed to determine the specificity and selectivity of each conjugate for FAP in vitro and in vivo. Results: FAP-targeted imaging and therapeutic conjugates showed high binding specificity and affinity in the low nanomolar range. Injection of FAP-targeted 99mTc into tumor-bearing mice enabled facile detection of tumor xenografts with little off-target uptake. Optical imaging of malignant lesions was also readily achieved following intravenous injection of FAP-targeted near-infrared fluorescent dye. Finally, systemic administration of a tubulysin B conjugate of FL promoted complete eradication of solid tumors with no evidence of gross toxicity to the animals. Conclusion: In view of the near absence of FAP on healthy cells, we conclude that targeting of FAP on cancer-associated fibroblasts can enable highly specific imaging and therapy of solid tumors.
Assuntos
Fibroblastos Associados a Câncer/metabolismo , Endopeptidases/metabolismo , Proteínas de Membrana/metabolismo , Microambiente Tumoral/fisiologia , Animais , Antineoplásicos/metabolismo , Fibroblastos Associados a Câncer/fisiologia , Linhagem Celular Tumoral , Endopeptidases/genética , Endopeptidases/fisiologia , Feminino , Fibroblastos/metabolismo , Corantes Fluorescentes/química , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Nus , Camundongos SCID , Imagem Óptica/métodos , Tomografia Computadorizada de Emissão de Fóton Único/métodosRESUMO
The ability to detect and visualize cellular events and their associated target biological analytes through use of cell-permeable profluorogenic probes is dependent on the availability of activatable probes that respond rapidly and selectively to target analytes by production of fluorescent reporting molecules whose excitation and emission energies span a broad range. Herein is described a new probe, DCM-Cys, that preferentially reacts with cysteine to form a dicyanomethylene-4H-pyran (DCM) reporter whose red-energy fluorescence can be stimulated by two-photon, near-infrared excitation so as to provide visualization of cysteine presence inside living human cells with a high signal-to-background ratio. These aforementioned characteristics and the ability of DCM-Cys to provide selective, nanomolar-level in vitro cysteine detection, as demonstrated by its lack of significant response to other thiols and potential interfering agents from biological environments, are attributed to the molecular designs of the DCM-Cys probe and DCM reporter. Attachment of an acryl moiety to the DCM reporter via a self-eliminating, electron-withdrawing benzyl alcohol-carbamate linker offers a probe having selective, sensitive reaction with cysteine to rapidly produce a reporter whose energies of excitation and emission (λabsreport = 480 nm, λemisreport = 640 nm) are red-shifted from those of the DCM-Cys probe (λabsprobe = 440 nm, λemisprobe = 550 nm), thereby leading to low background signal from abundant probe and a large signal from the resulting reporter of cysteine presence.
Assuntos
Cisteína/análise , Corantes Fluorescentes/química , Microscopia de Fluorescência por Excitação Multifotônica , Espectroscopia de Luz Próxima ao Infravermelho , Benzopiranos/química , Linhagem Celular Tumoral , Cisteína/química , Humanos , Razão Sinal-Ruído , Compostos de Sulfidrila/químicaRESUMO
Sorghum-based traditional fermented food was screened for potential probiotic lactic acid bacteria. The isolates were identified by biochemical, physiological and genetic methods. Species identification was done by 16s rRNA sequence analysis. The functional probiotic potential of the two Lactobacillus species viz., Lactobacillus plantarum (Lact. plantarum) and Lactobacillus pentosus (Lact. pentosus) was assessed by different standard parameters. The strains were tolerant to pH 2 for 1 h and resistant to methicillin, kanamycin, vancomycin and norfloxacin. Two (Lact. plantarum COORG-3 and Lact. pentosus COORG-8) out of eight isolates recorded the cell surface hydrophobicity to be 59.12 and 64.06%, respectively. All the strains showed tolerance to artificial duodenum juice (pH 2) for 3 h, positive for bile salt hydrolase test and negative for haemolytic test. The neutralized cell-free supernatant of the strains Lact. pentosus COORG-4, Lact. plantarum COORG-1, Lact. plantarum COORG-7, Lact. pentosus COORG-8 and Lact. plantarum COORG-3 showed good antibiofilm activity. Lact. pentosus COORG-8 exhibited 74% activity against Pseudomonas aeruginosa-MTCC 7903 and Lact. plantarum COORG-7 showed 68% inhibition of biofilm against Klebsiella pneumonia MTCC 7407. Three (Lact. plantarum COORG-7, Lact. pentosus COORG-5 and Lact. pentosus COORG 8) out of eight isolates exhibited a good antimicrobial activity against Listeria monocytogenes and five isolates (Lact. pentosus COORG 2, Lact. plantarum COORG 1, Lact. plantarum COORG 4, Lact. pentosus COORG 3 and Lact. plantarum COORG 6) are active against Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, Enterobacter aerogenes, Klebsiella pneumonia, Enterococcus faecalis. The study also evaluated the cholesterol lowering property of the Lactobacillus strains using hen egg yolk as the cholesterol source. The cholesterol in hen egg yolk was assimilated by 74.12 and 68.26% by Lact. plantarum COORG 4 and Lact. pentosus COORG 7, respectively. The results of the present study suggest that the Lactobacillus strains isolated and characterized from sorghum-based fermented product may be used as probiotic strains for therapeutic applications.
Assuntos
Lactobacillus plantarum/fisiologia , Lactobacillus/fisiologia , Probióticos , Sorghum/microbiologia , Antibacterianos/farmacologia , Bacillus subtilis/crescimento & desenvolvimento , Colesterol/análise , DNA Bacteriano/genética , Farmacorresistência Bacteriana Múltipla , Gema de Ovo/química , Gema de Ovo/microbiologia , Enterobacter aerogenes/crescimento & desenvolvimento , Enterococcus faecalis/crescimento & desenvolvimento , Escherichia coli/crescimento & desenvolvimento , Fermentação , Microbiologia de Alimentos , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Klebsiella pneumoniae/crescimento & desenvolvimento , Lactobacillus/efeitos dos fármacos , Lactobacillus/genética , Lactobacillus/isolamento & purificação , Lactobacillus plantarum/efeitos dos fármacos , Lactobacillus plantarum/genética , Lactobacillus plantarum/isolamento & purificação , Listeria monocytogenes/crescimento & desenvolvimento , Filogenia , Pseudomonas aeruginosa/crescimento & desenvolvimento , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Staphylococcus aureus/crescimento & desenvolvimentoRESUMO
There is a very limited number of existing probes whose fluorescence is turned on in the presence of the class of biological thiols made up of glutathione, cysteine, and homocysteine. The extant probes for this class of biological thiols commonly have poor aqueous solubility and long analyte response times, and they demand a very high probe/thiol ratio for decreased time of significant reporter signal generation; knowledge regarding their selectivity with respect to other sulfur-based analytes is unclear. Described here is a previously unreported photoinduced electron-transfer-quenched probe (HMBQ-Nap 1) that offers highly selective and rapid in vitro detection of this class of biologically important thiols at low concentrations and low probe/thiol ratio, and importantly, very rapid imaging of these biological thiols in human cells.
Assuntos
Corantes Fluorescentes/química , Compostos de Sulfidrila/análise , Linhagem Celular Tumoral , Transporte de Elétrons , Humanos , Espectroscopia de Ressonância Magnética , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
A frontier area in the development of activatable (turn-on) fluorescence-based probes is that concerned with rapid and selective stimulus triggering of probe activation so as to allow for biomarker identification and cellular imaging. The work here is concerned with a cloaked fluorophore composed of a reporter whose fluorescence is efficiently quenched by it being bound to an activatable trigger group through a novel self-immolative linker. Highly selective and rapid activation of the trigger group is achieved by chemical and enzymatic means that result in activated trigger group detachment from the self-immolative linker, with the latter subsequently cleaved from the reporter autonomously, thereby unmasking intense, red-shifted fluorescence emission. To achieve this success, we used a trimethyl-locked quinone propionic acid trigger group and an N-methyl-p-aminobenzyl alcohol self-immolative linker attached to the reporter. Delineated here are the synthesis and characterization of this cloaked fluorophore and the evaluation of its triggered turning on in the presence of an up-regulated enzyme in human cancer cells, NAD(P)H: quinone oxidoreductase-1 (NQO1, DT-diaphorase, EC 1.6.99.2).
Assuntos
Fluorescência , NAD(P)H Desidrogenase (Quinona)/metabolismo , Processos Fotoquímicos , Células HT29 , Humanos , Estrutura Molecular , NAD(P)H Desidrogenase (Quinona)/químicaRESUMO
NAD(P)H:quinone oxidoreductase type I (NQO1) is a target enzyme for triggered delivery of drugs at inflamed tissue and tumor sites, particularly those that challenge traditional therapies. Prodrugs, macromolecules, and molecular assemblies possessing trigger groups that can be cleaved by environmental stimuli are vehicles with the potential to yield active drug only at prescribed sites. Furthermore, quinone propionic acids (QPAs) covalently attached to prodrugs or liposome surfaces can be removed by application of a reductive trigger stimulus, such as that from NQO1; their rates of reductive activation should be tunable via QPA structure. We explored in detail the recombinant human NAD(P)H:quinone oxidoreductase type I (rhNQO1)-catalyzed NADH reduction of a family of substituted QPAs and obtained high precision kinetic parameters. It is found that small changes in QPA structure-in particular, single atom and function group substitutions on the quinone ring at R(1)-lead to significant impacts on the Michaelis constant (K(m)), maximum velocity (V(max)), catalytic constant (k(cat)), and catalytic efficiency (k(cat)/K(m)). Molecular docking simulations demonstrate that alterations in QPA structure result in large changes in QPA alignment and placement with respect to the flavin isoalloxazine ring in the active site of rhNQO1; a qualitative relationship exists between the kinetic parameters and the depth of QPA penetration into the rhNQO1 active site. From a quantitative perspective, a very good correlation is observed between log(k(cat)/K(m)) and the molecular-docking-derived distance between the flavin hydride donor site and quinone hydride acceptor site in the QPAs, an observation that is in agreement with developing theories. The comprehensive kinetic and molecular modeling knowledge obtained for the interaction of recombinant human NQO1 with the quinone propionic acid analogues provides insight into the design and implementation of the QPA trigger groups for drug delivery applications.