RESUMEN
Wet age-related macular degeneration (AMD), characterized by leaky neovessels emanating from the choroid, is a main cause of blindness. As current treatments for wet AMD require regular intravitreal injections of anti-vascular endothelial growth factor (VEGF) biologics, there is a need for the development of less invasive treatments. Here, we designed an allosteric inhibitor of end binding-3 (EB3) protein, termed EBIN, which reduces the effects of environmental stresses on endothelial cells by limiting pathological calcium signaling. Delivery of EBIN via eye drops in mouse and non-human primate (NHP) models of wet AMD prevents both neovascular leakage and choroidal neovascularization. EBIN reverses the epigenetic changes induced by environmental stresses, allowing an activation of a regenerative program within metabolic-active endothelial cells comprising choroidal neovascularization (CNV) lesions. These results suggest the therapeutic potential of EBIN in preventing the degenerative processes underlying wet AMD.
Asunto(s)
Neovascularización Coroidal , Degeneración Macular Húmeda , Ratones , Animales , Células Endoteliales/metabolismo , Neovascularización Coroidal/tratamiento farmacológico , Neovascularización Coroidal/metabolismo , Neovascularización Coroidal/patología , Degeneración Macular Húmeda/tratamiento farmacológico , Degeneración Macular Húmeda/metabolismoRESUMEN
End-binding proteins (EBs) associate with the growing microtubule plus ends to regulate microtubule dynamics as well as the interaction with intracellular structures. EB3 contributes to pathological vascular leakage through interacting with the inositol 1,4,5-trisphosphate receptor 3 (IP3R3), a calcium channel located at the endoplasmic reticulum membrane. The C-terminal domain of EB3 (residues 200-281) is functionally important for this interaction because it contains the effector binding sites, a prerequisite for EB3 activity and specificity. Structural data for this domain is limited. Here, we report the backbone chemical shift assignments for the human EB3 C-terminal domain and computationally explore its EB3 conformations. Backbone assignments, along with computational models, will allow future investigation of EB3 structural dynamics, interactions with effectors, and will facilitate the development of novel EB3 inhibitors.
Asunto(s)
Proteínas Asociadas a Microtúbulos/química , Resonancia Magnética Nuclear Biomolecular , Humanos , Modelos Moleculares , Dominios Proteicos , Estructura Secundaria de ProteínaRESUMEN
NMR allows assessment of protein structure in solution. Unlike conventional X-ray crystallography that provides snapshots of protein conformations, all conformational states are simultaneously accessible to analysis by NMR. This is a significant advantage for discovery and characterization of allosteric effects. These effects are observed when binding at one site of the protein affects another distinct site through conformational transitions. Allosteric regulation of proteins has been observed in multiple physiological processes in health and disease, providing an opportunity for the development of allosteric inhibitors. These compounds do not directly interact with the orthosteric site of the protein but influence its structure and function. In this book chapter, we provide an overview on how NMR methods are utilized to identify allosteric sites and to discover novel inhibitors, highlighting examples from the field. We also describe how NMR has contributed to understanding of allosteric mechanisms and propose that it is likely to play an important role in clarification and further development of key concepts of allostery.
Asunto(s)
Sitio Alostérico , Descubrimiento de Drogas , Ligandos , Espectroscopía de Resonancia Magnética , Regulación Alostérica , Sitios de Unión , Descubrimiento de Drogas/métodos , Descubrimiento de Drogas/tendencias , Conformación ProteicaRESUMEN
Protein tyrosine kinase 6 (PTK6, also called BRK) is overexpressed and activated in human prostate cancer. Loss of the tumor suppressor PTEN, a frequent event in prostate cancer, leads to PTK6 activation at the plasma membrane and its oncogenic signaling. The small molecule inhibitor vemurafenib, also known as PLX4032, and its tool analog PLX4720 were designed to inhibit constitutively active BRAF V600E, yet they also have potent effects against PTK6. Vemurafenib is used in the treatment of metastatic melanoma, but its efficacy in prostate cancer has not been assessed. When activated at the plasma membrane, PTK6 promotes signaling through FAK, EGFR, and ERK1/2, and we show this can be blocked by vemurafenib. In addition, PTK6-mediated cell growth, migration, and invasion are inhibited upon vemurafenib administration. Using a flank xenograft model, vemurafenib treatment reduced tumor burden. Using saturation transfer difference NMR and molecular docking, we demonstrate that vemurafenib binds in the active site of PTK6, inhibiting its activation. These structural studies provide insight into the PTK6-vemurafenib complex, which can be utilized for further refinement chemistry, whereas functional studies demonstrate that active PTK6 is a viable drug target in prostate cancer.
Asunto(s)
Proteínas de Neoplasias/química , Neoplasias de la Próstata/tratamiento farmacológico , Proteínas Tirosina Quinasas/química , Vemurafenib/farmacología , Animales , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Receptores ErbB/genética , Quinasa 1 de Adhesión Focal/genética , Xenoinjertos , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Ratones , Simulación del Acoplamiento Molecular , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Fosfohidrolasa PTEN/genética , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas B-raf/genética , Transducción de Señal/efectos de los fármacos , Vemurafenib/químicaRESUMEN
BACKGROUND: Chemokine signaling through CCR3 is a key regulatory pathway for eosinophil recruitment into tissues associated with allergic inflammation and asthma. To date, none of the CCR3 antagonists have shown efficacy in clinical trials. One reason might be their unbiased mode of inhibition that prevents receptor internalization, leading to drug tolerance. OBJECTIVE: We sought to develop a novel peptide nanoparticle CCR3 inhibitor (R321) with a biased mode of inhibition that would block G protein signaling but enable or promote receptor internalization. METHODS: Self-assembly of R321 peptide into nanoparticles and peptide binding to CCR3 were analyzed by means of dynamic light scattering and nuclear magnetic resonance. Inhibitory activity on CCR3 signaling was assessed in vitro by using flow cytometry, confocal microscopy, and Western blot analysis in a CCR3+ eosinophil cell line and blood eosinophils. In vivo effects of R321 were assessed by using a triple-allergen mouse asthma model. RESULTS: R321 self-assembles into nanoparticles and binds directly to CCR3, altering receptor function. Half-maximal inhibitory concentration values for eotaxin-induced chemotaxis of blood eosinophils are in the low nanomolar range. R321 inhibits only the early phase of extracellular signal-regulated kinase 1/2 activation and not the late phase generally associated with ß-arrestin recruitment and receptor endocytosis, promoting CCR3 internalization and degradation. In vivo R321 effectively blocks eosinophil recruitment into the blood, lungs, and airways and prevents airway hyperresponsiveness in a mouse eosinophilic asthma model. CONCLUSIONS: R321 is a potent and selective antagonist of the CCR3 signaling cascade. Inhibition through a biased mode of antagonism might hold significant therapeutic promise by eluding the formation of drug tolerance.
Asunto(s)
Eosinófilos/inmunología , Hipersensibilidad/tratamiento farmacológico , Pulmón/inmunología , Nanopartículas/uso terapéutico , Péptidos/uso terapéutico , Receptores CCR3/antagonistas & inhibidores , Hipersensibilidad Respiratoria/tratamiento farmacológico , Alérgenos/inmunología , Línea Celular , Movimiento Celular , Proteínas de Unión al GTP/antagonistas & inhibidores , Humanos , Espectroscopía de Resonancia Magnética , Unión Proteica , Transducción de SeñalRESUMEN
Repeated dosing of drugs targeting G protein-coupled receptors can stimulate antagonist tolerance, which reduces their efficacy; thus, strategies to avoid tolerance are needed. The efficacy of AMD3100, a competitive antagonist of the chemokine receptor CXCR4 that mobilizes leukemic blasts from the bone marrow into the blood to sensitize them to chemotherapy, is reduced after prolonged treatment. Tolerance to AMD3100 increases the abundance of CXCR4 on the surface of leukemic blasts, which promotes their rehoming to the bone marrow. AMD3100 inhibits both G protein signaling by CXCR4 and ß-arrestin1/2-dependent receptor endocytosis. We demonstrated that biased antagonists of G protein-dependent chemotaxis but not ß-arrestin1/2 recruitment and subsequent receptor endocytosis avoided tolerance. The peptide antagonist X4-2-6, which is derived from transmembrane helix 2 and extracellular loop 1 of CXCR4, limited chemotaxis and signaling but did not promote CXCR4 accumulation on the cell surface or cause tolerance. The activity of X4-2-6 was due to its distinct mechanism of inhibition of CXCR4. The peptide formed a ternary complex with the receptor and its ligand, the chemokine CXCL12. Within this complex, X4-2-6 released the portion of CXCL12 critical for receptor-mediated activation of G proteins but enabled the rest of the chemokine to recruit ß-arrestins to the receptor. In contrast, AMD3100 displaced all components of the chemokine responsible for CXCR4 activation. We further identified a small molecule with similar biased antagonist properties to those of X4-2-6, which may provide a viable alternative to patients when antagonist tolerance prevents drugs from reaching efficacy.
Asunto(s)
Tolerancia a Medicamentos , Proteínas de Unión al GTP/antagonistas & inhibidores , Receptores CXCR4/antagonistas & inhibidores , Receptores CXCR4/química , Transducción de Señal , Animales , Bencilaminas , Células CHO , Quimiocina CXCL12/metabolismo , Quimiotaxis , Cricetinae , Cricetulus , Ciclamas , Endocitosis , Fibroblastos/efectos de los fármacos , Compuestos Heterocíclicos/farmacología , Humanos , Células Jurkat , Ligandos , Ratones , Fosforilación , Dominios Proteicos , Células THP-1 , beta-Arrestina 1/metabolismo , Arrestina beta 2/metabolismoRESUMEN
Honokiol is a natural product and an emerging drug for a wide variety of malignancies, including hematopoietic malignancies, sarcomas, and common epithelial tumors. The broad range of activity of honokiol against numerous malignancies with diverse genetic backgrounds suggests that honokiol is inhibiting an activity that is common to multiple malignancies. Oncogenic transcription factor FOXM1 is one of the most overexpressed oncoproteins in human cancer. Here we found that honokiol inhibits FOXM1-mediated transcription and FOXM1 protein expression. More importantly, we found that honokiol's inhibitory effect on FOXM1 is a result of binding of honokiol to FOXM1. This binding is specific to honokiol, a dimerized allylphenol, and was not observed in compounds that either were monomeric allylphenols or un-substituted dihydroxy phenols. This indicates that both substitution and dimerization of allylphenols are required for physical interaction with FOXM1. We thus demonstrate a novel and specific mechanism for FOXM1 inhibition by honokiol, which partially may explain its anticancer activity in cancer cells.
Asunto(s)
Compuestos de Bifenilo/farmacología , Proteína Forkhead Box M1/antagonistas & inhibidores , Lignanos/farmacología , Animales , Compuestos de Bifenilo/química , Línea Celular Tumoral , Regulación hacia Abajo/efectos de los fármacos , Humanos , Lignanos/química , Ratones , Inhibidores de Proteasoma/farmacología , Activación Transcripcional/efectos de los fármacosRESUMEN
TP53-regulated inhibitor of apoptosis 1 (TRIAP1) is a novel apoptosis inhibitor that binds HSP70 in the cytoplasm and blocks the formation of the apoptosome and caspase-9 activation. TRIAP1 has been shown to be upregulated in many types of cancers; however, its role remains elusive. We determined the TRIAP1 mRNA levels in a panel of human tissues and found its expression to be ubiquitous. Normal breast, as well as non-tumorigenic breast cells, exhibited lower TRIAP1 mRNA levels than breast cancer cells or their drug-resistant derivatives. TRIAP1 is a small, evolutionarily conserved protein that is 76 amino acids long. We found that yeast cells, in which the TRIAP1 homologue was knocked out, had increased sensitivity to doxorubicin. Equally, RNA interference in breast cancer drug-resistant cells demonstrated that downregulation of TRIAP1 impaired cell growth in the presence of doxorubicin. As expected, caspase-9 activation was diminished after overexpression of TRIAP1 in drug-resistant cells. Importantly, stable transfections of a TRIAP1 expression plasmid in CAL51 cells led to a marked increase in the number of doxorubicin-resistant clones, that was abolished when cells expressed hairpins targeting TRIAP1. In addition, we showed that TRIAP1 expression was also triggered by estrogen deprivation in MCF-7 cells. Although both polyclonal and monoclonal antibodies generated for the present study failed to robustly detect TRIAP1, we demonstrated that TRIAP1 represents a novel marker for drug resistance in breast cancer cells and it may be used in the stratification of breast cancer patients once a suitable antibody has been developed. Equally, these studies open potential drug development strategies for blocking TRIAP1 activity and avoiding drug resistance.
Asunto(s)
Antibióticos Antineoplásicos/farmacología , Neoplasias de la Mama/genética , Doxorrubicina/farmacología , Resistencia a Antineoplásicos , Péptidos y Proteínas de Señalización Intracelular/genética , Apoptosis , Neoplasias de la Mama/tratamiento farmacológico , Caspasa 9/genética , Línea Celular Tumoral , Proliferación Celular , Femenino , Humanos , Células MCF-7 , ARN Interferente Pequeño/farmacología , Regulación hacia ArribaRESUMEN
K-Ras4B belongs to a family of small GTPases that regulates cell growth, differentiation and survival. K-ras is frequently mutated in cancer. K-Ras4B association with the plasma membrane through its farnesylated and positively charged C-terminal hypervariable region (HVR) is critical to its oncogenic function. However, the structural mechanisms of membrane association are not fully understood. Here, using confocal microscopy, surface plasmon resonance, and molecular dynamics simulations, we observed that K-Ras4B can be distributed in rigid and loosely packed membrane domains. Its membrane binding domain interaction with phospholipids is driven by membrane fluidity. The farnesyl group spontaneously inserts into the disordered lipid microdomains, whereas the rigid microdomains restrict the farnesyl group penetration. We speculate that the resulting farnesyl protrusion toward the cell interior allows oligomerization of the K-Ras4B membrane binding domain in rigid microdomains. Unlike other Ras isoforms, K-Ras4B HVR contains a single farnesyl modification and positively charged polylysine sequence. The high positive charge not only modulates specific HVR binding to anionic phospholipids but farnesyl membrane orientation. Phosphorylation of Ser-181 prohibits spontaneous farnesyl membrane insertion. The mechanism illuminates the roles of HVR modifications in K-Ras4B targeting microdomains of the plasma membrane and suggests an additional function for HVR in regulation of Ras signaling.