RESUMO
Macrocyclic drugs can address an increasing range of molecular targets but enabling central nervous system (CNS) access to these drugs has been viewed as an intractable problem. We designed and synthesized a series of quinolinium-modified cyclosporine derivatives targeted to the mitochondrial cyclophilin D protein. Modification of the cation to enable greater delocalization was confirmed by x-ray crystallography of the cations. Critically, greater delocalization improved brain concentrations. Assessment of the compounds in preclinical assays and for pharmacokinetics identified a molecule JP1-138 with at least 20 times the brain levels of a non-delocalized compound or those reported for cyclosporine. Levels were maintained over 24 hours together with low hERG potential. The paradigm outlined here could have widespread utility in the treatment of CNS diseases.
Assuntos
Compostos de Quinolínio , Animais , Humanos , Compostos de Quinolínio/química , Compostos de Quinolínio/farmacocinética , Ciclosporina/química , Ciclosporina/farmacocinética , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/efeitos dos fármacos , Cristalografia por Raios X , Peptídeos/química , Peptídeos/farmacocinética , Encéfalo/metabolismo , Encéfalo/efeitos dos fármacos , CamundongosRESUMO
Inflammation and fibrosis are important components of diseases that contribute to the malfunction of epithelia and endothelia. The Rho guanine nucleotide exchange factor (GEF) GEF-H1/ARHGEF-2 is induced in disease and stimulates inflammatory and fibrotic processes, cell migration, and metastasis. Here, we have generated peptide inhibitors to block the function of GEF-H1. Inhibitors were designed using a structural in silico approach or by isolating an inhibitory sequence from the autoregulatory C-terminal domain. Candidate inhibitors were tested for their ability to block RhoA/GEF-H1 binding in vitro, and their potency and specificity in cell-based assays. Successful inhibitors were then evaluated in models of TGFß-induced fibrosis, LPS-stimulated endothelial cell-cell junction disruption, and cell migration. Finally, the most potent inhibitor was successfully tested in an experimental retinal disease mouse model, in which it inhibited blood vessel leakage and ameliorated retinal inflammation when treatment was initiated after disease diagnosis. Thus, an antagonist that blocks GEF-H1 signaling effectively inhibits disease features in in vitro and in vivo disease models, demonstrating that GEF-H1 is an effective therapeutic target and establishing a new therapeutic approach.
Assuntos
Doenças Retinianas , Transdução de Sinais , Animais , Fibrose , Inflamação , Camundongos , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismoRESUMO
Vascular endothelial growth factors (VEGFs) are the key regulators of blood and lymphatic vessels' formation and function. Each of the proteins from the homologous family VEGFA, VEGFB, VEGFC and VEGFD employs a core cysteine-knot structural domain for the specific interaction with one or more of the cognate tyrosine kinase receptors. Additional diversity is exhibited by the involvement of neuropilins-transmembrane co-receptors, whose b1 domain contains the binding site for the C-terminal sequence of VEGFs. Although all relevant isoforms of VEGFs that interact with neuropilins contain the required C-terminal Arg residue, there is selectivity of neuropilins and VEGF receptors for the VEGF proteins, which is reflected in the physiological roles that they mediate. To decipher the contribution made by the C-terminal sequences of the individual VEGF proteins to that functional differentiation, we determined structures of molecular complexes of neuropilins and VEGF-derived peptides and examined binding interactions for all neuropilin-VEGF pairs experimentally and computationally. While X-ray crystal structures and ligand-binding experiments highlighted similarities between the ligands, the molecular dynamics simulations uncovered conformational preferences of VEGF-derived peptides beyond the C-terminal arginine that contribute to the ligand selectivity of neuropilins. The implications for the design of the selective antagonists of neuropilins' functions are discussed.
Assuntos
Neuropilinas , Fator A de Crescimento do Endotélio Vascular , Ligantes , Neuropilinas/química , Neuropilinas/genética , Neuropilinas/metabolismo , Peptídeos , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fatores de Crescimento do Endotélio VascularRESUMO
Vascular endothelial growth factors (VEGFs) regulate significant pathways in angiogenesis, myocardial and neuronal protection, metabolism, and cancer progression. The VEGF-B growth factor is involved in cell survival, anti-apoptotic and antioxidant mechanisms, through binding to VEGF receptor 1 and neuropilin-1 (NRP1). We employed surface plasmon resonance technology and X-ray crystallography to analyse the molecular basis of the interaction between VEGF-B and the b1 domain of NRP1, and developed VEGF-B C-terminus derived peptides to be used as chemical tools for studying VEGF-B - NRP1 related pathways. Peptide lipidation was used as a means to stabilise the peptides. VEGF-B-derived peptides containing a C-terminal arginine show potent binding to NRP1-b1. Peptide lipidation increased binding residence time and improved plasma stability. A crystal structure of a peptide with NRP1 demonstrated that VEGF-B peptides bind at the canonical C-terminal arginine binding site. VEGF-B C-terminus imparts higher affinity for NRP1 than the corresponding VEGF-A165 region. This tight binding may impact on the activity and selectivity of the full-length protein. The VEGF-B167 derived peptides were more effective than VEGF-A165 peptides in blocking functional phosphorylation events. Blockers of VEGF-B function have potential applications in diabetes and non-alcoholic fatty liver disease.
Assuntos
Neuropilina-1/metabolismo , Peptídeos/metabolismo , Fator B de Crescimento do Endotélio Vascular/metabolismo , Humanos , Neuropilina-1/química , Peptídeos/química , Ligação Proteica , Fator B de Crescimento do Endotélio Vascular/químicaRESUMO
Counteracting innate immunity is essential for successful viral replication. Host cyclophilins (Cyps) have been implicated in viral evasion of host antiviral responses, although the mechanisms are still unclear. Here, we show that hepatitis C virus (HCV) co-opts the host protein CypA to aid evasion of antiviral responses dependent on the effector protein kinase R (PKR). Pharmacological inhibition of CypA rescues PKR from antagonism by HCV NS5A, leading to activation of an interferon regulatory factor-1 (IRF1)-driven cell intrinsic antiviral program that inhibits viral replication. These findings further the understanding of the complexity of Cyp-virus interactions, provide mechanistic insight into the remarkably broad antiviral spectrum of Cyp inhibitors, and uncover novel aspects of PKR activity and regulation. Collectively, our study identifies a novel antiviral mechanism that harnesses cellular antiviral immunity to suppress viral replication.
Assuntos
Ciclofilina A/antagonistas & inibidores , Hepacivirus/fisiologia , Fator Regulador 1 de Interferon/imunologia , Proteínas não Estruturais Virais/metabolismo , Replicação Viral , eIF-2 Quinase/genética , Linhagem Celular Tumoral , Ciclofilina A/imunologia , Humanos , eIF-2 Quinase/imunologiaRESUMO
Multiple sclerosis (MS) is a major cause of disability in young adults. Following an unknown trigger (or triggers), the immune system attacks the myelin sheath surrounding axons, leading to progressive nerve cell death. Antibodies and small-molecule drugs directed against B cells have demonstrated good efficacy in slowing progression of the disease. This review focusses on small-molecule drugs that can affect B-cell biology and may have utility in disease management. The risk genes for MS are examined from the drug target perspective. Existing small-molecule therapies for MS with B-cell actions together with new drugs in development are described. The potential for experimental molecules with B-cell effects is also considered. Small molecules can have diverse actions on B cells and be cytotoxic, anti-inflammatory and anti-viral. The current B cell-directed therapies often kill B-cell subsets, which can be effective but lead to side effects and toxicity. A deeper understanding of B-cell biology and the effect on MS disease should lead to new drugs with better selectivity, efficacy, and an improved safety profile. Small-molecule drugs, once the patent term has expired, provide a uniquely sustainable form of healthcare.
Assuntos
Antineoplásicos , Linfócitos B , Esclerose Múltipla , Animais , Linfócitos B/fisiologia , Camundongos , Esclerose Múltipla/imunologia , Esclerose Múltipla/terapia , Bibliotecas de Moléculas PequenasRESUMO
Autophagy protease ATG4B is a key regulator of the LC3/GABARAP conjugation system required for autophagosome formation, maturation and closure. Members of the ATG4 and the LC3/GABARAP family have been implicated in various diseases including cancer, and targeting the ATG4B protease has been suggested as a potential therapeutic anti-cancer strategy. Recently, it has been demonstrated that ATG4B is regulated by multiple post-translational modifications, including phosphorylation and de-phosphorylation. In order to identify regulators of ATG4B activity, we optimized a cell-based luciferase assay based on ATG4B-dependent release of Gaussia luciferase. We applied this assay in a proof-of-concept small molecule compound screen and identified activating compounds that increase cellular ATG4B activity. Next, we performed a high-throughput screen to identify kinases and phosphatases that regulate cellular ATG4B activity using siRNA mediated knockdown and cDNA overexpression. Of these, we provide preliminary evidence that the kinase AKT2 enhances ATG4B activity in cells. We provide all raw and processed data from the screens as a resource for further analysis. Overall, our findings provide novel insights into the regulation of ATG4B and highlight the importance of post-translational modifications of ATG4B.
RESUMO
We report the design, synthesis, and biological evaluation of some potent small-molecule neuropilin-1 (NRP1) antagonists. NRP1 is implicated in the immune response to tumors, particularly in Treg cell fragility, required for PD1 checkpoint blockade. The design of these compounds was based on a previously identified compound EG00229. The design of these molecules was informed and supported by X-ray crystal structures. Compound 1 (EG01377) was identified as having properties suitable for further investigation. Compound 1 was then tested in several in vitro assays and was shown to have antiangiogenic, antimigratory, and antitumor effects. Remarkably, 1 was shown to be selective for NRP1 over the closely related protein NRP2. In purified Nrp1+, FoxP3+, and CD25+ populations of Tregs from mice, 1 was able to block a glioma-conditioned medium-induced increase in TGFß production. This comprehensive characterization of a small-molecule NRP1 antagonist provides the basis for future in vivo studies.
Assuntos
Imunomodulação/efeitos dos fármacos , Neuropilina-1/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/metabolismo , Fator de Crescimento Transformador beta/biossíntese , Inibidores da Angiogênese/química , Inibidores da Angiogênese/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Desenho de Fármacos , Humanos , Camundongos , Modelos Moleculares , Conformação Molecular , Ácidos Pentanoicos/química , Ácidos Pentanoicos/farmacologia , Bibliotecas de Moléculas Pequenas/química , Linfócitos T Reguladores/imunologia , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
Primary effusion lymphoma (PEL) is a lymphogenic disorder associated with Kaposi's sarcoma-associated herpesvirus (KSHV) infection. Key to the survival and proliferation of PEL is the canonical NF-κB pathway, which becomes constitutively activated following overexpression of the viral oncoprotein KSHV vFLIP (ks-vFLIP). This arises from its capacity to form a complex with the modulatory subunit of the IκB kinase (IKK) kinase, IKKγ (or NEMO), resulting in the overproduction of proteins that promote cellular survival and prevent apoptosis, both of which are important drivers of tumorigenesis. Using a combination of cell-based and biophysical assays together with structural techniques, we showed that the observed resistance to cell death is largely independent of autophagy or major death receptor signaling pathways and demonstrated that direct targeting of the ks-vFLIP-IKKγ interaction both in cells and in vitro can be achieved using IKKγ-mimetic peptides. Our results further reveal that these peptides not only induce cell killing but also potently sensitize PEL to the proapoptotic agents tumor necrosis factor alpha and etoposide and are the first to confirm ks-vFLIP as a tractable target for the treatment of PEL and related disorders.IMPORTANCE KSHV vFLIP (ks-vFLIP) has been shown to have a crucial role in cellular transformation, in which it is vital for the survival and proliferation of primary effusion lymphoma (PEL), an aggressive malignancy associated with infection that is resistant to the majority of chemotherapeutic drugs. It operates via subversion of the canonical NF-κB pathway, which requires a physical interaction between ks-vFLIP and the IKK kinase modulatory subunit IKKγ. While this interaction has been directly linked to protection against apoptosis, it is unclear whether the suppression of other cell death pathways implicated in ks-vFLIP pathogenesis is an additional contributor. We demonstrate that the interaction between ks-vFLIP and IKKγ is pivotal in conferring resistance to apoptosis. Additionally, we show that the ks-vFLIP-IKKγ complex can be disrupted using peptides leading to direct killing and the sensitization of PEL cells to proapoptotic agents. Our studies thus provide a framework for future therapeutic interventions.
Assuntos
Apoptose , Herpesvirus Humano 8/fisiologia , Quinase I-kappa B/química , Peptídeos/metabolismo , Peptídeos/farmacologia , Sarcoma de Kaposi/virologia , Autofagia , Etoposídeo/farmacologia , Herpesvirus Humano 8/química , Humanos , Quinase I-kappa B/metabolismo , Células Jurkat , Mimetismo Molecular , Peptídeos/química , Ligação Proteica , Sarcoma de Kaposi/fisiopatologia , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Proteínas Virais/metabolismoRESUMO
BACKGROUND AND PURPOSE: Cystic fibrosis (CF) is a debilitating disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which codes for a Cl-/HCO3 - channel. F508del, the most common CF-associated mutation, causes both gating and biogenesis defects in the CFTR protein. This paper describes the optimization of two fluorescence assays, capable of measuring CFTR function and cellular localization, and their use in a pilot drug screen. EXPERIMENTAL APPROACH: HEK293 cells expressing YFP-F508del-CFTR, in which halide sensitive YFP is tagged to the N-terminal of CFTR, were used to screen a small library of compounds based on the VX-770 scaffold. Cells expressing F508del-CFTR-pHTomato, in which a pH sensor is tagged to the fourth extracellular loop of CFTR, were used to measure CFTR plasma membrane exposure following chronic treatment with the novel potentiators. KEY RESULTS: Active compounds with efficacy ~50% of VX-770, micromolar potency, and structurally distinct from VX-770 were identified in the screen. The F508del-CFTR-pHTomato assay suggests that the hit compound MS131A, unlike VX-770, does not decrease membrane exposure of F508del-CFTR. CONCLUSIONS AND IMPLICATIONS: Most known potentiators have a negative influence on F508del-CFTR biogenesis/stability, which means membrane exposure needs to be monitored early during the development of drugs targeting CFTR. The combined use of the two fluorescence assays described here provides a useful tool for the identification of improved potentiators and correctors. The assays could also prove useful for basic scientific investigations on F508del-CFTR, and other CF-causing mutations.
Assuntos
Aminofenóis/análise , Aminofenóis/farmacologia , Proteínas de Bactérias/análise , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Fluorescência , Proteínas Luminescentes/análise , Quinolonas/análise , Quinolonas/farmacologia , Bibliotecas de Moléculas Pequenas/análise , Bibliotecas de Moléculas Pequenas/farmacologia , Aminofenóis/síntese química , Aminofenóis/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Células HEK293 , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Estrutura Molecular , Quinolonas/síntese química , Quinolonas/química , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/químicaRESUMO
The mitochondrial permeability transition pore is a recognized drug target for neurodegenerative conditions such as multiple sclerosis and for ischemia-reperfusion injury in the brain and heart. The peptidylprolyl isomerase, cyclophilin D (CypD, PPIF), is a positive regulator of the pore, and genetic down-regulation or knock-out improves outcomes in disease models. Current inhibitors of peptidylprolyl isomerases show no selectivity between the tightly conserved cyclophilin paralogs and exhibit significant off-target effects, immunosuppression, and toxicity. We therefore designed and synthesized a new mitochondrially targeted CypD inhibitor, JW47, using a quinolinium cation tethered to cyclosporine. X-ray analysis was used to validate the design concept, and biological evaluation revealed selective cellular inhibition of CypD and the permeability transition pore with reduced cellular toxicity compared with cyclosporine. In an experimental autoimmune encephalomyelitis disease model of neurodegeneration in multiple sclerosis, JW47 demonstrated significant protection of axons and improved motor assessments with minimal immunosuppression. These findings suggest that selective CypD inhibition may represent a viable therapeutic strategy for MS and identify quinolinium as a mitochondrial targeting group for in vivo use.
Assuntos
Córtex Cerebral/efeitos dos fármacos , Ciclofilinas/antagonistas & inibidores , Proteínas de Transporte da Membrana Mitocondrial/antagonistas & inibidores , Esclerose Múltipla/prevenção & controle , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/uso terapêutico , Compostos de Quinolínio/uso terapêutico , Substituição de Aminoácidos , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Córtex Cerebral/imunologia , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Peptidil-Prolil Isomerase F , Ciclofilinas/genética , Ciclofilinas/metabolismo , Ciclosporinas/efeitos adversos , Ciclosporinas/síntese química , Ciclosporinas/farmacologia , Ciclosporinas/uso terapêutico , Células Hep G2 , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos Endogâmicos , Camundongos Knockout , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Esclerose Múltipla/imunologia , Esclerose Múltipla/metabolismo , Esclerose Múltipla/patologia , Mutação , Neurônios/imunologia , Neurônios/metabolismo , Neurônios/patologia , Fármacos Neuroprotetores/efeitos adversos , Fármacos Neuroprotetores/farmacologia , Peptídeos Cíclicos/efeitos adversos , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/farmacologia , Peptídeos Cíclicos/uso terapêutico , Compostos de Quinolínio/efeitos adversos , Compostos de Quinolínio/síntese química , Compostos de Quinolínio/farmacologia , Distribuição Aleatória , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/patologiaRESUMO
The interaction between VEGF-A and its neuropilin (NRP) receptors mediates a number of important biological effects. NRP1 and the related molecule NRP2 are widely expressed on multiple tumour types and throughout the tumour vasculature, and are emerging as critical molecules required for the progression of angiogenic diseases. Given the increasing evidence supporting a role for NRP1 in tumour development, there is growing interest in developing inhibitors of NRP1 interactions with VEGF and its other ligands. In order to probe the interaction we synthesised a number of exon 7- and 8-derived bicyclic peptides with N-terminal lipophilic groups and found a simple N-octanoyl derivative (EG00086) to be the most potent and functionally active. Detailed modelling studies indicated that new intramolecular hydrogen bonds were formed, stabilising the structure and possibly contributing to the potency. Removal of a salt bridge between D142 and R164 implicated in VEGF-A binding to neuropilin-1 had a minor effect on potency. Isothermal calorimetry was used to assess binding of EG00086 to NRP1 and NRP2, and the stability of the peptide in serum and in vivo was investigated. EG00086 is a potent blocker of VEGF-promoted cellular adhesion to extracellular matrices, and phosphorylation of p130Cas contributes to this effect.
Assuntos
Neuropilina-1/metabolismo , Peptídeos Cíclicos/química , Peptídeos Cíclicos/metabolismo , Fator A de Crescimento do Endotélio Vascular/química , Fator A de Crescimento do Endotélio Vascular/metabolismo , Sítios de Ligação , Adesão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Proteína Substrato Associada a Crk/metabolismo , Éxons/genética , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Humanos , Lipopeptídeos/química , Lipopeptídeos/metabolismo , Lipopeptídeos/farmacologia , Simulação de Dinâmica Molecular , Neuropilina-1/química , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/farmacologia , Fosforilação/efeitos dos fármacos , Ligação Proteica , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
OBJECTIVES: To identify and to characterize small-molecule inhibitors that target the subunit polymerization of the type 1 pilus assembly in uropathogenic Escherichia coli (UPEC). METHODS: Using an SDS-PAGE-based assay, in silico pre-filtered small-molecule compounds were screened for specific inhibitory activity against the critical subunit polymerization step of the chaperone-usher pathway during pilus biogenesis. The biological activity of one of the compounds was validated in assays monitoring UPEC type 1 pilus biogenesis, type 1 pilus-dependent biofilm formation and adherence to human bladder epithelial cells. The time dependence of the in vivo inhibitory activity and the overall effect of the compound on UPEC growth were determined. RESULTS: N-(4-chloro-phenyl)-2-{5-[4-(pyrrolidine-1-sulfonyl)-phenyl]-[1,3,4]oxadiazol-2-yl sulfanyl}-acetamide (AL1) inhibited in vitro pilus subunit polymerization. In bacterial cultures, AL1 disrupted UPEC type 1 pilus biogenesis and pilus-dependent biofilm formation, and resulted in the reduction of bacterial adherence to human bladder epithelial cells, without affecting bacterial cell growth. Bacterial exposure to the inhibitor led to an almost instantaneous loss of type 1 pili. CONCLUSIONS: We have identified and characterized a small molecule that interferes with the assembly of type 1 pili. The molecule targets the polymerization step during the subunit incorporation cycle of the chaperone-usher pathway. Our discovery provides new insight into the design and development of novel anti-virulence therapies targeting key virulence factors of bacterial pathogens.
Assuntos
Antibacterianos/farmacologia , Aderência Bacteriana/efeitos dos fármacos , Fímbrias Bacterianas/efeitos dos fármacos , Substâncias Macromoleculares/metabolismo , Multimerização Proteica/efeitos dos fármacos , Subunidades Proteicas/metabolismo , Escherichia coli Uropatogênica/efeitos dos fármacos , Animais , Biofilmes/efeitos dos fármacos , Linhagem Celular , Células Epiteliais/microbiologia , Humanos , Escherichia coli Uropatogênica/fisiologiaRESUMO
Human immunodeficiency virus (HIV)-1 is able to replicate in primary human macrophages without stimulating innate immunity despite reverse transcription of genomic RNA into double-stranded DNA, an activity that might be expected to trigger innate pattern recognition receptors. We reasoned that if correctly orchestrated HIV-1 uncoating and nuclear entry is important for evasion of innate sensors then manipulation of specific interactions between HIV-1 capsid and host factors that putatively regulate these processes should trigger pattern recognition receptors and stimulate type 1 interferon (IFN) secretion. Here we show that HIV-1 capsid mutants N74D and P90A, which are impaired for interaction with cofactors cleavage and polyadenylation specificity factor subunit 6 (CPSF6) and cyclophilins (Nup358 and CypA), respectively, cannot replicate in primary human monocyte-derived macrophages because they trigger innate sensors leading to nuclear translocation of NF-κB and IRF3, the production of soluble type 1 IFN and induction of an antiviral state. Depletion of CPSF6 with short hairpin RNA expression allows wild-type virus to trigger innate sensors and IFN production. In each case, suppressed replication is rescued by IFN-receptor blockade, demonstrating a role for IFN in restriction. IFN production is dependent on viral reverse transcription but not integration, indicating that a viral reverse transcription product comprises the HIV-1 pathogen-associated molecular pattern. Finally, we show that we can pharmacologically induce wild-type HIV-1 infection to stimulate IFN secretion and an antiviral state using a non-immunosuppressive cyclosporine analogue. We conclude that HIV-1 has evolved to use CPSF6 and cyclophilins to cloak its replication, allowing evasion of innate immune sensors and induction of a cell-autonomous innate immune response in primary human macrophages.
Assuntos
HIV-1/imunologia , Evasão da Resposta Imune , Imunidade Inata/imunologia , Macrófagos/imunologia , Macrófagos/virologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Ciclofilinas/metabolismo , Ciclosporina/metabolismo , Infecções por HIV/imunologia , Infecções por HIV/metabolismo , Infecções por HIV/patologia , Infecções por HIV/virologia , HIV-1/metabolismo , Humanos , Fator Regulador 3 de Interferon/metabolismo , Interferon Tipo I/imunologia , Interferon Tipo I/metabolismo , Macrófagos/citologia , Macrófagos/patologia , Chaperonas Moleculares/metabolismo , Monócitos/citologia , NF-kappa B/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Receptores de Reconhecimento de Padrão , Internalização do Vírus , Replicação Viral/imunologia , Fatores de Poliadenilação e Clivagem de mRNA/deficiência , Fatores de Poliadenilação e Clivagem de mRNA/genética , Fatores de Poliadenilação e Clivagem de mRNA/metabolismoRESUMO
In this study, avidin-biotin technology was combined with a multifunctional drug carrier modality i.e. liposomes to achieve an active and versatile targeting approach. The anti-cancer drug doxorubicin (DOX) was modified with direct biotinylation (B-DOX) (Allart et al., 2003), or encapsulated in biotinylated sterically stabilized pH-sensitive liposomes (BL-DOX), and targeted to the lentiviral vector transduced cells expressing an avidin fusion protein on the cell membrane (Lehtolainen et al., 2003; Lesch et al., 2009). The direct biotinylation of doxorubicin improved cell internalization in rat glioma (BT4C) cells expressing avidin fusion protein receptor but cell toxicity was reduced by 78-fold due to impaired nuclear localization. In contrast, liposomal formulations restored the biological activity of the DOX in several cell lines. However, mainly due to uptake via non-specific pathways the active targeting of BL-DOX was negligible in both in vitro and in vivo studies. Active targeting with multifunctional drug carrier systems is challenging and further studies will be needed to optimize the properties of targeted drug carrier and receptor expression systems.
Assuntos
Antibióticos Antineoplásicos/administração & dosagem , Avidina/administração & dosagem , Biotina/administração & dosagem , Doxorrubicina/administração & dosagem , Animais , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/farmacocinética , Avidina/genética , Biotina/genética , Biotinilação , Linhagem Celular Tumoral , Doxorrubicina/química , Doxorrubicina/farmacocinética , Humanos , Cinética , Lipossomos , Camundongos , Camundongos Nus , Ratos , Proteínas Recombinantes de Fusão/administração & dosagem , Distribuição TecidualRESUMO
The small molecule carrier class of biomolecule transporters, modeled on the third helix of the Antennapedia homeodomain, has previously been shown to transport active proteins into cells. Here, we show an improved synthetic route to small molecule carriers, including Molander chemistry using trifluoroborate salts to improve the yield of the Suzuki-Miyaura coupling step for the formation of the biphenyl backbone. The required boronic acids could be formed by the reaction of a 2-(dimethylamino)ethyl ether-modified aryl Grignard reagent with triisopropyl borate. The potential for the use of small molecule carriers as oligonucleotide-transporting agents was also explored by characterizing the interactions between small molecule carriers and siRNA. Molecular dynamics and NMR analysis indicated that the small molecule carrier guanidines are stabilized by π-cation interactions with the biphenyl system, thus not only increasing the basicity or pKa but also shielding the charge. The binding affinities of various small molecule carriers for siRNA were investigated using isothermal calorimetry and gel shift assays. Small molecule carrier-mediated siRNA delivery to cultured fibroblasts is demonstrated, showing that small molecule carriers possess the ability to transport functional siRNA into cells. Knockdown of Cdc7 kinase, a target for cancer, is achieved.
Assuntos
RNA Interferente Pequeno/química , Bibliotecas de Moléculas Pequenas/química , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Guanidina/química , Humanos , Cinética , Microscopia Confocal , Simulação de Dinâmica Molecular , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Bibliotecas de Moléculas Pequenas/síntese químicaRESUMO
We report the molecular design and synthesis of EG00229, 2, the first small molecule ligand for the VEGF-A receptor neuropilin 1 (NRP1) and the structural characterization of NRP1-ligand complexes by NMR spectroscopy and X-ray crystallography. Mutagenesis studies localized VEGF-A binding in the NRP1 b1 domain and a peptide fragment of VEGF-A was shown to bind at the same site by NMR, providing the basis for small molecule design. Compound 2 demonstrated inhibition of VEGF-A binding to NRP1 and attenuated VEGFR2 phosphorylation in endothelial cells. Inhibition of migration of endothelial cells was also observed. The viability of A549 lung carcinoma cells was reduced by 2, and it increased the potency of the cytotoxic agents paclitaxel and 5-fluorouracil when given in combination. These studies provide the basis for design of specific small molecule inhibitors of ligand binding to NRP1.
Assuntos
Antineoplásicos/síntese química , Neuropilina-1/fisiologia , Fragmentos de Peptídeos/síntese química , Fator A de Crescimento do Endotélio Vascular/fisiologia , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cristalografia por Raios X , Humanos , Concentração Inibidora 50 , Espectroscopia de Ressonância Magnética , Mutagênese Sítio-Dirigida , Neuropilina-1/antagonistas & inibidores , Neuropilina-1/ultraestrutura , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/farmacologia , Fragmentos de Peptídeos/ultraestrutura , Fosforilação , Relação Estrutura-Atividade , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/ultraestruturaRESUMO
The transducing ability of the third helix of transcription factor homeodomains is effectively mimicked by a biphenyl system displaying guanidine groups. The biphenyl class of small molecule carriers (SMoCs) can carry biomolecules into a wide variety of cell types. A "combinatorial" approach to the synthesis of SMoCs is described using sequential Pd(0) coupling chemistry to assemble the molecules from highly functionalized building blocks. SMoCs coupled to the DNA licensing repressor protein geminin can inhibit DNA replication in vitro. We conducted a structure-activity investigation utilizing a range of SMoC-geminin conjugates and demonstrate that both electrostatic and structural features are important for efficient uptake and functional activity. The best analogue was more efficient than either (Arg)(4) or (Arg)(8) linked to geminin. Effective inhibition of DNA synthesis was achieved in fibroblasts and osteosarcoma cell lines.
Assuntos
Materiais Biomiméticos/síntese química , Materiais Biomiméticos/farmacologia , Compostos de Bifenilo/síntese química , Compostos de Bifenilo/farmacologia , Células/metabolismo , Paládio/química , Arginina/química , Benzeno/química , Materiais Biomiméticos/química , Materiais Biomiméticos/metabolismo , Compostos de Bifenilo/química , Compostos de Bifenilo/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Replicação do DNA/efeitos dos fármacos , Citometria de Fluxo , Humanos , Hidrocarbonetos Halogenados/química , Inibidores da Síntese de Ácido Nucleico/síntese química , Inibidores da Síntese de Ácido Nucleico/química , Inibidores da Síntese de Ácido Nucleico/metabolismo , Inibidores da Síntese de Ácido Nucleico/farmacologia , Eletricidade Estática , Relação Estrutura-AtividadeRESUMO
We designed and synthesized small-molecule mimics of an alpha-helical peptide protein transduction domain (PTD). These small-molecule carriers, which we termed SMoCs, are easily coupled to biomolecules, and efficiently deliver dye molecules and recombinant proteins into a variety of cell types. We designed the SMoCs using molecular modeling techniques. As an example of a protein cargo, we applied this new technology to the internalization of the DNA replication licensing repressor geminin, in vitro, providing evidence that extracellularly delivered SMoC-geminin can have an antiproliferative effect on human cancer cells. Uptake of SMoC-geminin was inhibited at 4 degrees C and by chlorpromazine, a compound that induces misassembly of clathrin-coated pits at the cell surface. Thus the mechanism of uptake is likely to be clathrin-mediated endocytosis.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Mimetismo Molecular , Transporte Proteico , Animais , Proteína do Homeodomínio de Antennapedia/química , Compostos de Bifenilo , Linhagem Celular Tumoral , Células Cultivadas , Clatrina/metabolismo , Invaginações Revestidas da Membrana Celular/química , Corantes/química , Corantes/metabolismo , Endocitose , Geminina , Células HeLa , Humanos , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Células NIH 3T3 , Peptídeos/química , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
Neuropilin-1 (NP-1) is a receptor for vascular endothelial growth factor-A165 (VEGF-A165) in endothelial cells. To define the role of NP-1 in the biological functions of VEGF, we developed a specific peptide antagonist of VEGF binding to NP-1 based on the NP-1 binding site located in the exon 7- and 8-encoded VEGF-A165 domain. The bicyclic peptide, EG3287, potently (K(i) 1.2 microM) and effectively (>95% inhibition at 100 microM) inhibited VEGF-A165 binding to porcine aortic endothelial cells expressing NP-1 (PAE/NP-1) and breast carcinoma cells expressing only NP-1 receptors for VEGF-A, but had no effect on binding to PAE/KDR or PAE/Flt-1. Molecular dynamics calculations, a nuclear magnetic resonance structure of EG3287, and determination of stability in media, indicated that it constitutes a stable subdomain very similar to the corresponding region of native VEGF-A165. The C terminus encoded by exon 8 and the three-dimensional structure were both critical for EG3287 inhibition of NP-1 binding, whereas modifications at the N terminus had little effect. Although EG3287 had no direct effect on VEGF-A165 binding to KDR receptors, it inhibited cross-linking of VEGF-A165 to KDR in human umbilical vein endothelial cells co-expressing NP-1, and inhibited stimulation of KDR and PLC-gamma tyrosine phosphorylation, activation of ERKs1/2 and prostanoid production. These findings characterize the first specific antagonist of VEGF-A165 binding to NP-1 and demonstrate that NP-1 is essential for optimum KDR activation and intracellular signaling. The results also identify a key role for the C-terminal exon 8 domain in VEGF-A165 binding to NP-1.