RESUMEN
Inflammatory responses mediated by NOD2 rely on RIP2 kinase and ubiquitin ligase XIAP for the activation of nuclear factor κB (NF-κB), mitogen-activated protein kinases (MAPKs), and cytokine production. Herein, we demonstrate that selective XIAP antagonism blocks NOD2-mediated inflammatory signaling and cytokine production by interfering with XIAP-RIP2 binding, which removes XIAP from its ubiquitination substrate RIP2. We also establish that the kinase activity of RIP2 is dispensable for NOD2 signaling. Rather, the conformation of the RIP2 kinase domain functions to regulate binding to the XIAP-BIR2 domain. Effective RIP2 kinase inhibitors block NOD2 signaling by disrupting RIP2-XIAP interaction. Finally, we identify NOD2 signaling and XIAP-dependent ubiquitination sites on RIP2 and show that mutating these lysine residues adversely affects NOD2 pathway signaling. Overall, these results reveal a critical role for the XIAP-RIP2 interaction in NOD2 inflammatory signaling and provide a molecular basis for the design of innovative therapeutic strategies based on XIAP antagonists and RIP2 kinase inhibitors.
Asunto(s)
Aminoquinolinas/farmacología , Inflamación/prevención & control , Proteína Adaptadora de Señalización NOD2/antagonistas & inhibidores , Dominios y Motivos de Interacción de Proteínas/efectos de los fármacos , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/metabolismo , Sulfonas/farmacología , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismo , Animales , Células Cultivadas , Humanos , Inflamación/metabolismo , Inflamación/patología , Ratones Endogámicos C57BL , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteína Adaptadora de Señalización NOD2/metabolismo , Fosforilación , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/antagonistas & inhibidores , Transducción de Señal , Ubiquitina/metabolismo , Ubiquitinación , Proteína Inhibidora de la Apoptosis Ligada a X/antagonistas & inhibidoresRESUMEN
The G2019S variant of LRRK2, which causes an increase in kinase activity, is associated with the occurrence of Parkinson's disease (PD). Potent, mutation-selective, and brain penetrant inhibitors of LRRK2 can suppress the biological effects specific to G2019S-LRRK2 that cause pathogenicity. We report the discovery of a series of cyanoindane and cyanotetralin kinase inhibitors culminating in compound 34 that demonstrated selective inhibition of phosphorylation of LRRK2 in the mouse brain. These novel inhibitors may further enable the precision medicine path for future PD therapeutics.
RESUMEN
The prosurvival BCL-2 family protein BCL-X(L) is often overexpressed in solid tumors and renders malignant tumor cells resistant to anticancer therapeutics. Enhancing apoptotic responses by inhibiting BCL-X(L) will most likely have widespread utility in cancer treatment and, instead of inhibiting multiple prosurvival BCL-2 family members, a BCL-X(L)-selective inhibitor would be expected to minimize the toxicity to normal tissues. We describe the use of a high-throughput screen to discover a new series of small molecules targeting BCL-X(L) and their structure-guided development by medicinal chemistry. The optimized compound, WEHI-539 (7), has high affinity (subnanomolar) and selectivity for BCL-X(L) and potently kills cells by selectively antagonizing its prosurvival activity. WEHI-539 will be an invaluable tool for distinguishing the roles of BCL-X(L) from those of its prosurvival relatives, both in normal cells and notably in malignant tumor cells, many of which may prove to rely upon BCL-X(L) for their sustained growth.
Asunto(s)
Antineoplásicos/farmacología , Diseño de Fármacos , Proteína bcl-X/antagonistas & inhibidores , Proteína bcl-X/química , Animales , Apoptosis , Benzotiazoles/química , Línea Celular Tumoral , Cristalografía por Rayos X , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Hidrazonas/química , Cinética , Ratones , Modelos Químicos , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Unión Proteica , Proteínas Proto-Oncogénicas c-bcl-2/genéticaRESUMEN
Proapoptotic receptor agonists cause cellular demise through the activation of the extrinsic and intrinsic apoptotic pathways. Inhibitor of apoptosis (IAP) proteins block apoptosis induced by diverse stimuli. Here, we demonstrate that IAP antagonists in combination with Fas ligand (FasL) or the death receptor 5 (DR5) agonist antibody synergistically stimulate death in cancer cells and inhibit tumor growth. Single-agent activity of IAP antagonists relies on tumor necrosis factor-alpha signaling. By contrast, blockade of tumor necrosis factor-alpha does not affect the synergistic activity of IAP antagonists with FasL or DR5 agonist antibody. In most cancer cells, proapoptotic receptor agonist-induced cell death depends on amplifying the apoptotic signal via caspase-8-mediated activation of Bid and subsequent activation of the caspase-9-dependent mitochondrial apoptotic pathway. In the investigated cancer cell lines, induction of apoptosis by FasL or DR5 agonist antibody can be inhibited by knockdown of Bid. However, knockdown of X chromosome-linked IAP (XIAP) or antagonism of XIAP allows FasL or DR5 agonist antibody to induce activation of effector caspases efficiently without the need for mitochondrial amplification of the apoptotic signal and thus rescues the effect of Bid knockdown in these cells.
Asunto(s)
Apoptosis/fisiología , Muerte Celular/fisiología , Proteína Ligando Fas/metabolismo , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/agonistas , Proteína Inhibidora de la Apoptosis Ligada a X/antagonistas & inhibidores , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismo , Animales , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/genética , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/metabolismo , Caspasas/metabolismo , Línea Celular Tumoral , Etanercept , Humanos , Inmunoglobulina G/genética , Inmunoglobulina G/metabolismo , Ratones , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Receptores del Factor de Necrosis Tumoral/genética , Receptores del Factor de Necrosis Tumoral/metabolismo , Transducción de Señal/fisiología , Trasplante Heterólogo , Factor de Necrosis Tumoral alfa/metabolismo , Proteína Inhibidora de la Apoptosis Ligada a X/genéticaRESUMEN
Interactions between Bcl-2 homology-3 (BH3)-only proteins and their pro-survival Bcl-2 family binding partners initiate the intrinsic apoptosis pathway. These interactions are mediated by a short helical motif, the BH3 domain, on the BH3-only protein, which inserts into a hydrophobic groove on the pro-survival molecule. To identify novel peptidic ligands that bind Mcl-1, a pro-survival protein relative of Bcl-2, both human and mouse Mcl-1 were screened against large randomized phage-displayed peptide libraries. We identified a number of 16-mer peptides with sub-micromolar affinity that were highly selective for Mcl-1, as well as being somewhat selective for the species of Mcl-1 (human or mouse) against which the library was panned. Interestingly, these sequences all strongly resembled natural BH3 domain sequences. By switching residues within the best of the human Mcl-1-binding sequences, or extending beyond the core sequence identified, we were able to alter the pro-survival protein interaction profile of this peptide such that it now bound all members tightly and was a potent killer when introduced into cells. Introduction of an amide lock constraint within this sequence also increased its helicity and binding to pro-survival proteins. These data provide new insights into the determinants of BH3 domain:pro-survival protein affinity and selectivity.
Asunto(s)
Péptidos/química , Péptidos/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Humanos , Ratones , Datos de Secuencia Molecular , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Biblioteca de Péptidos , Péptidos/síntesis química , Péptidos/genética , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-bcl-2/química , Proteínas Proto-Oncogénicas c-bcl-2/genética , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
A family of anti-apoptotic regulators known as IAP (inhibitor of apoptosis) proteins interact with multiple cellular partners and inhibit apoptosis induced by a variety of stimuli. c-IAP (cellular IAP) 1 and 2 are recruited to TNFR1 (tumour necrosis factor receptor 1)-associated signalling complexes, where they mediate receptor-induced NF-kappaB (nuclear factor kappaB) activation. Additionally, through their E3 ubiquitin ligase activities, c-IAP1 and c-IAP2 promote proteasomal degradation of NIK (NF-kappaB-inducing kinase) and regulate the non-canonical NF-kappaB pathway. In the present paper, we describe a novel ubiquitin-binding domain of IAPs. The UBA (ubiquitin-associated) domain of IAPs is located between the BIR (baculovirus IAP repeat) domains and the CARD (caspase activation and recruitment domain) or the RING (really interesting new gene) domain of c-IAP1 and c-IAP2 or XIAP (X-linked IAP) respectively. The c-IAP1 UBA domain binds mono-ubiquitin and Lys(48)- and Lys(63)-linked polyubiquitin chains with low-micromolar affinities as determined by surface plasmon resonance or isothermal titration calorimetry. NMR analysis of the c-IAP1 UBA domain-ubiquitin interaction reveals that this UBA domain binds the classical hydrophobic patch surrounding Ile(44) of ubiquitin. Mutations of critical amino acid residues in the highly conserved MGF (Met-Gly-Phe) binding loop of the UBA domain completely abrogate ubiquitin binding. These mutations in the UBA domain do not overtly affect the ubiquitin ligase activity of c-IAP1 or the participation of c-IAP1 and c-IAP2 in the TNFR1 signalling complex. Treatment of cells with IAP antagonists leads to proteasomal degradation of c-IAP1 and c-IAP2. Deletion or mutation of the UBA domain decreases this degradation, probably by diminishing the interaction of the c-IAPs with the proteasome. These results suggest that ubiquitin binding may be an important mechanism for rapid turnover of auto-ubiquitinated c-IAP1 and c-IAP2.
Asunto(s)
Proteínas Inhibidoras de la Apoptosis/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitina/metabolismo , Secuencia de Aminoácidos , Sitios de Unión/genética , Calorimetría , Línea Celular , Línea Celular Tumoral , Dicroismo Circular , Humanos , Proteínas Inhibidoras de la Apoptosis/antagonistas & inhibidores , Proteínas Inhibidoras de la Apoptosis/química , Cinética , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Datos de Secuencia Molecular , FN-kappa B/metabolismo , Poliubiquitina/metabolismo , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Homología de Secuencia de Aminoácido , Resonancia por Plasmón de Superficie , Ubiquitinación , Quinasa de Factor Nuclear kappa BRESUMEN
Pathogenic variants in the leucine-rich repeat kinase 2 (LRRK2) gene have been identified that increase the risk for developing Parkinson's disease in a dominantly inherited fashion. These pathogenic variants, of which G2019S is the most common, cause abnormally high kinase activity, and compounds that inhibit this activity are being pursued as potentially disease-modifying therapeutics. Because LRRK2 regulates important cellular processes, developing inhibitors that can selectively target the pathogenic variant while sparing normal LRRK2 activity could offer potential advantages in heterozygous carriers. We conducted a high-throughput screen and identified a single selective compound that preferentially inhibited G2019S-LRRK2. Optimization of this scaffold led to a series of novel, potent, and highly selective G2019S-LRRK2 inhibitors.
Asunto(s)
Indazoles/farmacología , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Tetrazoles/farmacología , Animales , Células HEK293 , Ensayos Analíticos de Alto Rendimiento , Humanos , Indazoles/síntesis química , Indazoles/farmacocinética , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Ratones , Estructura Molecular , Mutación , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacocinética , Pirimidinas/síntesis química , Pirimidinas/farmacocinética , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/farmacocinética , Bibliotecas de Moléculas Pequeñas/farmacología , Relación Estructura-Actividad , Tetrazoles/síntesis química , Tetrazoles/farmacocinéticaRESUMEN
Apoptosis is deregulated in most, if not all, cancers, including hematological malignancies. Smac mimetics that antagonize Inhibitor of Apoptosis (IAP) proteins have so far largely been investigated in acute myeloid leukemia (AML) cell lines; however, little is yet known on the therapeutic potential of Smac mimetics in primary AML samples. In this study, we therefore investigated the antileukemic activity of the Smac mimetic BV6 in diagnostic samples of 67 adult AML patients and correlated the response to clinical, cytogenetic and molecular markers and gene expression profiles. Treatment with cytarabine (ara-C) was used as a standard chemotherapeutic agent. Interestingly, about half (51%) of primary AML samples are sensitive to BV6 and 21% intermediate responsive, while 28% are resistant. Notably, 69% of ara-C-resistant samples show a good to fair response to BV6. Furthermore, combination treatment with ara-C and BV6 exerts additive effects in most samples. Whole-genome gene expression profiling identifies cell death, TNFR1 and NF-κB signaling among the top pathways that are activated by BV6 in BV6-sensitive, but not in BV6-resistant cases. Furthermore, sensitivity of primary AML blasts to BV6 correlates with significantly elevated expression levels of TNF and lower levels of XIAP in diagnostic samples, as well as with NPM1 mutation. In a large set of primary AML samples, these data provide novel insights into factors regulating Smac mimetic response in AML and have important implications for the development of Smac mimetic-based therapies and related diagnostics in AML.
Asunto(s)
Muerte Celular/efectos de los fármacos , Regulación Leucémica de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular/farmacología , Leucemia Mieloide Aguda/tratamiento farmacológico , Proteínas Mitocondriales/farmacología , Adulto , Anciano , Anciano de 80 o más Años , Apoptosis , Proteínas Reguladoras de la Apoptosis , Línea Celular Tumoral , Supervivencia Celular , Citarabina/farmacología , Femenino , Perfilación de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Mutación , FN-kappa B/metabolismo , Nucleofosmina , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Transducción de Señal , Resultado del Tratamiento , Adulto JovenRESUMEN
Cellular inhibitor of apoptosis 1 (cIAP1) is a ubiquitin ligase with critical roles in the control of programmed cell death and NF-κB signaling. Under normal conditions, the protein exists as an autoinhibited monomer, but proapoptotic signals lead to its dimerization, activation and proteasomal degradation. This view of cIAP1 as a binary switch has been informed by static structural studies that cannot access the protein's dynamics. Here, we use NMR spectroscopy to study micro- and millisecond motions of specific domain interfaces in human cIAP1 and use time-resolved small-angle X-ray scattering to observe the global conformational changes necessary for activation. Although motions within each interface of the 'closed' monomer are insufficient to activate cIAP1, they enable associations with catalytic partners and activation factors. We propose that these internal motions facilitate rapid peptide-induced opening and dimerization of cIAP1, which undergoes a dramatic spring-loaded structural transition.
Asunto(s)
Proteínas Inhibidoras de la Apoptosis/química , Proteínas Inhibidoras de la Apoptosis/metabolismo , Humanos , Cinética , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Conformación Proteica , Multimerización de Proteína , Estructura Terciaria de Proteína , Dispersión del Ángulo Pequeño , Ubiquitina/metabolismo , Enzimas Activadoras de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas , Difracción de Rayos XRESUMEN
A-1155463, a highly potent and selective BCL-XL inhibitor, was discovered through nuclear magnetic resonance (NMR) fragment screening and structure-based design. This compound is substantially more potent against BCL-XL-dependent cell lines relative to our recently reported inhibitor, WEHI-539, while possessing none of its inherent pharmaceutical liabilities. A-1155463 caused a mechanism-based and reversible thrombocytopenia in mice and inhibited H146 small cell lung cancer xenograft tumor growth in vivo following multiple doses. A-1155463 thus represents an excellent tool molecule for studying BCL-XL biology as well as a productive lead structure for further optimization.
RESUMEN
An attractive approach for developing therapeutic peptides is to enhance binding to their targets by stabilizing their α-helical conformation, for example, stabilized BimBH3 peptides (BimSAHB) designed to induce apoptosis. Unexpectedly, we found that such modified peptides have reduced affinity for their targets, the pro-survival Bcl-2 proteins. We attribute this loss in affinity to disruption of a network of stabilizing intramolecular interactions present in the bound state of the native peptide. Altering this network may compromise binding affinity, as in the case of the BimBH3 stapled peptide studied here. Moreover, cells exposed to these peptides do not readily undergo apoptosis, strongly indicating that BimSAHB is not inherently cell permeable.
Asunto(s)
Apoptosis , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/metabolismo , Animales , Humanos , Ratones , Modelos Moleculares , Estructura Secundaria de ProteínaRESUMEN
Tumor necrosis factor (TNF) family members are essential for the development and proper functioning of the immune system. TNF receptor (TNFR) signaling is mediated through the assembly of protein signaling complexes that activate the nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) pathways in a ubiquitin-dependent manner. The cellular inhibitor of apoptosis (c-IAP) proteins c-IAP1 and c-IAP2 are E3 ubiquitin ligases that are recruited to TNFR signaling complexes through their constitutive association with the adaptor protein TNFR-associated factor 2 (TRAF2). We demonstrated that c-IAP1 and c-IAP2 were required for canonical activation of NF-κB and MAPK by members of the TNFR family. c-IAPs were required for the recruitment of inhibitor of κB kinase ß (IKKß), the IKK regulatory subunit NF-κB essential modulator (NEMO), and RBCK1/Hoil1-interacting protein (HOIP) to TNFR signaling complexes and the induction of gene expression by TNF family members. In contrast, TNFRs that stimulated the noncanonical NF-κB pathway triggered translocation of c-IAPs, TRAF2, and TRAF3 from the cytosol to membrane fractions, which led to their proteasomal and lysosomal degradation. Finally, we established that signaling by B cell-activating factor receptor 3 induced the cytosolic depletion of TRAF3, which enabled noncanonical NF-κB activation. These results define c-IAP proteins as critical regulators of the activation of NF-κB and MAPK signaling pathways by members of the TNFR superfamily.
Asunto(s)
Proteínas Inhibidoras de la Apoptosis/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Transducción de Señal/inmunología , Factores de Necrosis Tumoral/metabolismo , Western Blotting , Proteínas Portadoras/metabolismo , Línea Celular Tumoral , Silenciador del Gen , Humanos , Quinasa I-kappa B/metabolismo , Proteínas Inhibidoras de la Apoptosis/inmunología , Transporte de Proteínas , ARN Interferente Pequeño/genética , Receptores de Interleucina-4/metabolismo , Factor 2 Asociado a Receptor de TNF/metabolismo , Factores de Necrosis Tumoral/inmunología , Ubiquitina/metabolismo , Ubiquitina-Proteína LigasasRESUMEN
A series of compounds were designed and synthesized as antagonists of cIAP1/2, ML-IAP, and XIAP based on the N-terminus, AVPI, of mature Smac. Compound 1 (GDC-0152) has the best profile of these compounds; it binds to the XIAP BIR3 domain, the BIR domain of ML-IAP, and the BIR3 domains of cIAP1 and cIAP2 with K(i) values of 28, 14, 17, and 43 nM, respectively. These compounds promote degradation of cIAP1, induce activation of caspase-3/7, and lead to decreased viability of breast cancer cells without affecting normal mammary epithelial cells. Compound 1 inhibits tumor growth when dosed orally in the MDA-MB-231 breast cancer xenograft model. Compound 1 was advanced to human clinical trials, and it exhibited linear pharmacokinetics over the dose range (0.049 to 1.48 mg/kg) tested. Mean plasma clearance in humans was 9 ± 3 mL/min/kg, and the volume of distribution was 0.6 ± 0.2 L/kg.
Asunto(s)
Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Proteínas Inhibidoras de la Apoptosis/antagonistas & inhibidores , Tiadiazoles/síntesis química , Tiadiazoles/farmacología , Animales , Antineoplásicos/química , Antineoplásicos/farmacocinética , Apoptosis/efectos de los fármacos , Proteína 3 que Contiene Repeticiones IAP de Baculovirus , Unión Competitiva , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Caspasas/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Ensayos Clínicos Fase I como Asunto , Femenino , Humanos , Proteínas Inhibidoras de la Apoptosis/metabolismo , Masculino , Tiadiazoles/química , Tiadiazoles/farmacocinética , Ubiquitina-Proteína LigasasRESUMEN
Defects in apoptosis contribute to treatment resistance and poor outcome of pancreatic cancer, calling for novel therapeutic strategies. Here, we provide the first evidence that nuclear factor (NF) κB is required for Smac mimetic-mediated sensitization of pancreatic carcinoma cells for gemcitabine-induced apoptosis. The Smac mimetic BV6 cooperates with gemcitabine to reduce cell viability and to induce apoptosis. In addition, BV6 significantly enhances the cytotoxicity of several anticancer drugs against pancreatic carcinoma cells, including doxorubicin, cisplatin, and 5-fluorouracil. Molecular studies reveal that BV6 stimulates NF-κB activation, which is further increased in the presence of gemcitabine. Importantly, inhibition of NF-κB by overexpression of the dominant-negative IκBα superrepressor significantly decreases BV6- and gemcitabine-induced apoptosis, demonstrating that NF-κB exerts a proapoptotic function in this model of apoptosis. In support of this notion, inhibition of tumor necrosis factor α (TNFα) by the TNFα blocking antibody Enbrel reduces BV6- and gemcitabine-induced activation of caspase 8 and 3, loss of mitochondrial membrane potential, and apoptosis. By demonstrating that BV6 and gemcitabine trigger a NF-κB-dependent, TNFα-mediated loop to activate apoptosis signaling pathways and caspase-dependent apoptotic cell death, our findings have important implications for the development of Smac mimetic-based combination protocols in the treatment of pancreatic cancer.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Desoxicitidina/análogos & derivados , Péptidos y Proteínas de Señalización Intracelular/química , Proteínas Mitocondriales/química , Imitación Molecular , FN-kappa B/metabolismo , Neoplasias Pancreáticas/metabolismo , Animales , Antineoplásicos/administración & dosificación , Proteínas Reguladoras de la Apoptosis , Caspasas/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Desoxicitidina/administración & dosificación , Desoxicitidina/farmacología , Humanos , Ratones , Factor de Necrosis Tumoral alfa/metabolismo , GemcitabinaRESUMEN
Limitations to the application of molecularly targeted cancer therapies are the inability to accurately match patient with effective treatment and the absence of a prompt readout of posttreatment response. Noninvasive agents that rapidly report vascular endothelial growth factor (VEGF) levels using positron emission tomography (PET) have the potential to enhance anti-angiogenesis therapies. Using phage display, two distinct classes of peptides were identified that bind to VEGF with nanomolar affinity and high selectivity. Co-crystal structures of these different peptide classes demonstrate that both bind to the receptor-binding region of VEGF. (18)F-radiolabelling of these peptides facilitated the acquisition of PET images of tumor VEGF levels in a HM7 xenograph model. The images obtained from one 59-residue probe, (18)F-Z-3B, 2 hr postinjection are comparable to those obtained with anti-VEGF antibody B20 72 hr postinjection. Furthermore, VEGF levels in growing SKOV3 tumors were followed using (18)F-Z-3B as a PET probe with VEGF levels increasing with tumor size.
Asunto(s)
Neoplasias/diagnóstico por imagen , Péptidos/química , Péptidos/metabolismo , Tomografía de Emisión de Positrones/métodos , Factor A de Crecimiento Endotelial Vascular/análisis , Factor A de Crecimiento Endotelial Vascular/metabolismo , Secuencia de Aminoácidos , Animales , Química Clic , Cristalografía por Rayos X , Humanos , Ratones , Ratones Desnudos , Modelos Moleculares , Datos de Secuencia Molecular , Neoplasias/metabolismo , Biblioteca de Péptidos , Péptidos/síntesis química , Unión Proteica , Multimerización de Proteína , Factor A de Crecimiento Endotelial Vascular/químicaRESUMEN
Searching for new strategies to bypass apoptosis resistance, we investigated the potential of the Smac mimetic BV6 in Jurkat leukemia cells deficient in key molecules of the death receptor pathway. Here, we demonstrate for the first time that Smac mimetic primes apoptosis-resistant, FADD- or caspase-8-deficient leukemia cells for TNFα-induced necroptosis in a synergistic manner. In contrast to TNFα, Smac mimetic significantly enhances CD95-induced apoptosis in wild-type but not in FADD-deficient cells. Interestingly, Smac mimetic- and TNFα-mediated cell death occurs without characteristic features of apoptosis (i.e., caspase activation, DNA fragmentation) in FADD-deficient cells. By comparison, Smac mimetic and TNFα trigger activation of caspase-8, -9, and -3 and DNA fragmentation in wild-type cells. Consistently, the caspase inhibitor zVAD.fmk fails to block Smac mimetic- and TNFα-triggered cell death in FADD- or caspase-8-deficient cells, while it confers protection in wild-type cells. By comparison, necrostatin-1, an RIP1 kinase inhibitor, abolishes Smac mimetic- and TNFα-induced cell death in FADD- or caspase-8-deficient. Thus, Smac mimetic enhances TNFα-induced cell death in leukemia cells via two distinct pathways in a context-dependent manner: it primes apoptosis-resistant cells lacking FADD or caspase-8 to TNFα-induced, RIP1-dependent and caspase-independent necroptosis, whereas it sensitizes apoptosis-proficient cells to TNFα-mediated, caspase-dependent apoptosis. These findings have important implications for the therapeutic exploitation of necroptosis as an alternative cell death program to overcome apoptosis resistance.
Asunto(s)
Apoptosis/efectos de los fármacos , Materiales Biomiméticos/farmacología , Caspasa 8/metabolismo , Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Proteínas Reguladoras de la Apoptosis , Western Blotting , Caspasa 3/metabolismo , Caspasa 9/metabolismo , Fragmentación del ADN/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Sinergismo Farmacológico , Activación Enzimática/efectos de los fármacos , Proteína de Dominio de Muerte Asociada a Fas/deficiencia , Humanos , Imidazoles/farmacología , Indoles/farmacología , Proteínas Inhibidoras de la Apoptosis/antagonistas & inhibidores , Proteínas Inhibidoras de la Apoptosis/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Células Jurkat , Microscopía Fluorescente , Proteínas Mitocondriales/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas de Unión al ARN/metabolismoRESUMEN
Inhibitor of apoptosis (IAP) proteins are negative regulators of cell death. IAP family members contain RING domains that impart E3 ubiquitin ligase activity. Binding of endogenous or small-molecule antagonists to select baculovirus IAP repeat (BIR) domains within cellular IAP (cIAP) proteins promotes autoubiquitination and proteasomal degradation and so releases inhibition of apoptosis mediated by cIAP. Although the molecular details of antagonist-BIR domain interactions are well understood, it is not clear how this binding event influences the activity of the RING domain. Here biochemical and structural studies reveal that the unliganded, multidomain cIAP1 sequesters the RING domain within a compact, monomeric structure that prevents RING dimerization. Antagonist binding induces conformational rearrangements that enable RING dimerization and formation of the active E3 ligase.
Asunto(s)
Proteínas Inhibidoras de la Apoptosis/antagonistas & inhibidores , Proteínas Inhibidoras de la Apoptosis/química , Secuencia de Aminoácidos , Animales , Línea Celular , Línea Celular Tumoral , Clonación Molecular , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas Inhibidoras de la Apoptosis/metabolismo , Ratones , Modelos Biológicos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Complejo de la Endopetidasa Proteasomal/metabolismo , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Estructura Secundaria de Proteína , Dispersión del Ángulo Pequeño , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Ubiquitinadas/química , Proteínas Ubiquitinadas/metabolismo , UbiquitinaciónRESUMEN
The inhibitor of apoptosis (IAP) proteins are critical regulators of cancer cell survival, which makes them attractive targets for therapeutic intervention in cancers. Herein, we describe the structure-based design of IAP antagonists with high affinities and selectivity (>2000-fold) for c-IAP1 over XIAP and their functional characterization as activators of apoptosis in tumor cells. Although capable of inducing cell death and preventing clonogenic survival, c-IAP-selective antagonists are significantly less potent in promoting apoptosis when compared to pan-selective compounds. However, both pan-IAP- and c-IAP-selective antagonists stimulate c-IAP1 and c-IAP2 degradation and activation of NF-kappaB pathways with comparable potencies. Therefore, although compounds that specifically target c-IAP1 and c-IAP2 are capable of inducing apoptosis, antagonism of the c-IAP proteins and XIAP is required for efficient induction of cancer cell death by IAP antagonists.
Asunto(s)
Proteínas Inhibidoras de la Apoptosis/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Proteína Inhibidora de la Apoptosis Ligada a X/antagonistas & inhibidores , Sitios de Unión , Muerte Celular/efectos de los fármacos , Línea Celular , Humanos , Proteínas Inhibidoras de la Apoptosis/química , Proteínas Inhibidoras de la Apoptosis/metabolismo , Melanoma/patología , Modelos Moleculares , Estructura Molecular , FN-kappa B/metabolismo , Unión Proteica , Relación Estructura-ActividadRESUMEN
The inhibitor of apoptosis (IAP) proteins are a family of anti-apoptotic regulators found in viruses and metazoans. c-IAP1 and c-IAP2 are recruited to tumor necrosis factor receptor 1 (TNFR1)-associated complexes where they can regulate receptor-mediated signaling. Both c-IAP1 and c-IAP2 have been implicated in TNFalpha-stimulated NF-kappaB activation. However, individual c-IAP1 and c-IAP2 gene knock-outs in mice did not reveal changes in TNF signaling pathways, and the phenotype of a combined deficiency of c-IAPs has yet to be reported. Here we investigate the role of c-IAP1 and c-IAP2 in TNFalpha-stimulated activation of NF-kappaB. We demonstrate that TNFalpha-induced NF-kappaB activation is severely diminished in the absence of both c-IAP proteins. In addition, combined absence of c-IAP1 and c-IAP2 rendered cells sensitive to TNFalpha-induced cell death. Using cells with genetic ablation of c-IAP1 or cells where the c-IAP proteins were eliminated using IAP antagonists, we show that TNFalpha-induced RIP1 ubiquitination is abrogated in the absence of c-IAPs. Furthermore, we reconstitute the ubiquitination process with purified components in vitro and demonstrate that c-IAP1, in collaboration with the ubiquitin conjugating enzyme (E2) enzyme UbcH5a, mediates polymerization of Lys-63-linked chains on RIP1. Therefore, c-IAP1 and c-IAP2 are required for TNFalpha-stimulated RIP1 ubiquitination and NF-kappaB activation.