RESUMEN
AB598 is a CD39 inhibitory antibody being pursued for the treatment of solid tumors in combination with chemotherapy and immunotherapy. CD39 metabolizes extracellular ATP (eATP), an alarmin capable of promoting anti-tumor immune responses, into adenosine, an immuno-inhibitory metabolite. By inhibiting CD39, the consumption of eATP is reduced, resulting in a pro-inflammatory milieu in which eATP can activate myeloid cells to promote anti-tumor immunity. The preclinical characterization of AB598 provides a mechanistic rationale for combining AB598 with chemotherapy in the clinic. Chemotherapy can induce ATP release from tumor cells and, when preserved by AB598, both chemotherapy-induced eATP and exogenously added ATP promote the function of monocyte-derived dendritic cells via P2Y11 signaling. Inhibition of CD39 in the presence of ATP can promote inflammasome activation in in vitro-derived macrophages, an effect mediated by P2X7. In a MOLP8 murine xenograft model, AB598 results in full inhibition of intratumoral enzymatic activity, an increase in intratumoral ATP, a decrease of extracellular CD39 on tumor cells, and ultimately, control of tumor growth. In cynomolgus monkeys, systemically dosed AB598 results in effective enzymatic inhibition in tissues, full peripheral and tissue target engagement, and a reduction in cell surface CD39 both in tissues and in the periphery. Taken together, these data support a promising therapeutic strategy of harnessing the eATP generated by standard-of-care chemotherapies to prime the tumor microenvironment for a productive anti-tumor immune response.
RESUMEN
Lysine specific demethylase 1 (LSD1) acts as an epigenetic eraser by specifically demethylating mono- and histone 3 lysine 4 (H3K4) and H3 lysine 9 (H3K9) residues. LSD1 has been pursued as a promising therapeutic target for the treatment of human cancer, and a number of LSD1 inhibitors have been advanced into clinical development. In the present study, we describe our discovery of pyrrolo[2,3-c]pyridines as a new class of highly potent and reversible LSD1 inhibitors, designed on the basis of a previously reported LSD1 inhibitor GSK-354. Among them, 46 shows an IC50 value of 3.1 nM in inhibition of LSD1 enzymatic activity and inhibits cell growth with IC50 values of 0.6 nM in the MV4;11 acute leukemia cell line and 1.1 nM in the H1417 small-cell lung cancer cell line. Compound 46 (LSD1-UM-109) is a novel, highly potent, and reversible LSD1 inhibitor and serves as a promising lead compound for further optimization.
RESUMEN
Multi-tyrosine kinase inhibitors (MTKIs) have thus far had limited success in the treatment of castration-resistant prostate cancer (CRPC). Here, we report a phase I-cleared orally bioavailable MTKI, ESK981, with a novel autophagy inhibitory property that decreased tumor growth in diverse preclinical models of CRPC. The anti-tumor activity of ESK981 was maximized in immunocompetent tumor environments where it upregulated CXCL10 expression through the interferon gamma pathway and promoted functional T cell infiltration, which resulted in enhanced therapeutic response to immune checkpoint blockade. Mechanistically, we identify the lipid kinase PIKfyve as the direct target of ESK981. PIKfyve-knockdown recapitulated ESK981's anti-tumor activity and enhanced the therapeutic benefit of immune checkpoint blockade. Our study reveals that targeting PIKfyve via ESK981 turns tumors from cold into hot through inhibition of autophagy, which may prime the tumor immune microenvironment in advanced prostate cancer patients and be an effective treatment strategy alone or in combination with immunotherapies.
Asunto(s)
Inhibidores de Puntos de Control Inmunológico , Neoplasias de la Próstata Resistentes a la Castración , Autofagia , Humanos , Inmunoterapia/métodos , Masculino , Fosfatidilinositol 3-Quinasas/farmacología , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Microambiente TumoralRESUMEN
Inhibition of embryonic ectoderm development (EED) is a new cancer therapeutic strategy. Herein, we report our discovery of EEDi-5285 as an exceptionally potent, efficacious, and orally active EED inhibitor. EEDi-5285 binds to the EED protein with an IC50 value of 0.2 nM and inhibits cell growth with IC50 values of 20 pM and 0.5 nM in the Pfeiffer and KARPAS422 lymphoma cell lines, respectively, carrying an EZH2 mutation. EEDi-5285 is approximately 100 times more potent than EED226 in binding to EED and >300 times more potent than EED226 in inhibition of cell growth in the KARPAS422 cell line. EEDi-5285 has excellent pharmacokinetics and achieves complete and durable tumor regression in the KARPAS422 xenograft model in mice with oral administration. The cocrystal structure of EEDi-5285 in a complex with EED defines the precise structural basis for their high binding affinity. EEDi-5285 is the most potent and efficacious EED inhibitor reported to date.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Complejo Represivo Polycomb 2/antagonistas & inhibidores , Complejo Represivo Polycomb 2/química , Bibliotecas de Moléculas Pequeñas/farmacología , Administración Oral , Animales , Antineoplásicos/administración & dosificación , Disponibilidad Biológica , Línea Celular Tumoral , Cristalografía por Rayos X , Histonas/metabolismo , Humanos , Linfoma/tratamiento farmacológico , Linfoma/patología , Ratones SCID , Complejo Represivo Polycomb 2/metabolismo , Bibliotecas de Moléculas Pequeñas/administración & dosificación , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Androgen receptor (AR) antagonists, such as enzalutamide, have had a major impact on the treatment of metastatic castration-resistant prostate cancer (CRPC). However, even with the advent of AR antagonist therapies, patients continue to develop resistance, and new strategies to combat continued AR signalling are needed. Here, we develop AR degraders using PROteolysis TArgeting Chimeric (PROTAC) technology in order to determine whether depletion of AR protein can overcome mechanisms of resistance commonly associated with current AR-targeting therapies. ARD-61 is the most potent of the AR degraders and effectively induces on-target AR degradation with a mechanism consistent with the PROTAC design. Compared to clinically-approved AR antagonists, administration of ARD-61 in vitro and in vivo results in more potent anti-proliferative, pro-apoptotic effects and attenuation of downstream AR target gene expression in prostate cancer cells. Importantly, we demonstrate that ARD-61 functions in enzalutamide-resistant model systems, characterized by diverse proposed mechanisms of resistance that include AR amplification/overexpression, AR mutation, and expression of AR splice variants, such as AR-V7. While AR degraders are unable to bind and degrade AR-V7, they continue to inhibit tumor cell growth in models overexpressing AR-V7. To further explore this, we developed several isogenic prostate cell line models in which AR-V7 is highly expressed, which also failed to influence the cell inhibitory effects of AR degraders, suggesting that AR-V7 is not a functional resistance mechanism for AR antagonism. These data provide compelling evidence that full-length AR remains a prominent oncogenic driver of prostate cancers which have developed resistance to AR antagonists and highlight the clinical potential of AR degraders for treatment of CRPC.
Asunto(s)
Antagonistas de Receptores Androgénicos/farmacología , Andrógenos/genética , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Receptores Androgénicos/efectos de los fármacos , Andrógenos/metabolismo , Animales , Benzamidas/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Ratones , Nitrilos/farmacología , Feniltiohidantoína/farmacología , Neoplasias de la Próstata Resistentes a la Castración/genética , Neoplasias de la Próstata Resistentes a la Castración/patología , Receptores Androgénicos/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Inhibition of the menin-mixed lineage leukemia (MLL) protein-protein interaction is a promising new therapeutic strategy for the treatment of acute leukemia carrying MLL fusion (MLL leukemia). We describe herein our structure-based design, synthesis, and evaluation of a new class of small-molecule inhibitors of the menin-MLL interaction (hereafter called menin inhibitors). Our efforts have resulted in the discovery of highly potent menin inhibitors, as exemplified by compound 42 (M-89). M-89 binds to menin with a Kd value of 1.4 nM and effectively engages cellular menin protein at low nanomolar concentrations. M-89 inhibits cell growth in the MV4;11 and MOLM-13 leukemia cell lines carrying MLL fusion with IC50 values of 25 and 55 nM, respectively, and demonstrates >100-fold selectivity over the HL-60 leukemia cell line lacking MLL fusion. The determination of a co-crystal structure of M-89 in a complex with menin provides the structural basis for their high-affinity interaction. Further optimization of M-89 may lead to a new class of therapy for the treatment of MLL leukemia.
Asunto(s)
Descubrimiento de Drogas/métodos , Leucemia Mieloide/tratamiento farmacológico , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Enfermedad Aguda , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Células HL-60 , Humanos , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patología , Modelos Químicos , Estructura Molecular , Proteína de la Leucemia Mieloide-Linfoide/química , Unión Proteica/efectos de los fármacos , Proteínas Proto-Oncogénicas/química , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-ActividadRESUMEN
We report herein the discovery of highly potent PROTAC degraders of androgen receptor (AR), as exemplified by compound 34 (ARD-69). ARD-69 induces degradation of AR protein in AR-positive prostate cancer cell lines in a dose- and time-dependent manner. ARD-69 achieves DC50 values of 0.86, 0.76, and 10.4 nM in LNCaP, VCaP, and 22Rv1 AR+ prostate cancer cell lines, respectively. ARD-69 is capable of reducing the AR protein level by >95% in these prostate cancer cell lines and effectively suppressing AR-regulated gene expression. ARD-69 potently inhibits cell growth in these AR-positive prostate cancer cell lines and is >100 times more potent than AR antagonists. A single dose of ARD-69 effectively reduces the level of AR protein in xenograft tumor tissue in mice. Further optimization of ARD-69 may ultimately lead to a new therapy for AR+, castration-resistant prostate cancer.
Asunto(s)
Antagonistas de Receptores Androgénicos/química , Proteolisis , Receptores Androgénicos/metabolismo , Antagonistas de Receptores Androgénicos/farmacología , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Expresión Génica/efectos de los fármacos , Humanos , Ligandos , Masculino , Ratones , Ratones SCID , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Unión Proteica , Proteolisis/efectos de los fármacos , Receptores Androgénicos/genética , Relación Estructura-Actividad , Trasplante Heterólogo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/química , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismoRESUMEN
Proteins of the bromodomain and extra-terminal (BET) family are epigenetics "readers" and promising therapeutic targets for cancer and other human diseases. We describe herein a structure-guided design of [1,4]oxazepines as a new class of BET inhibitors and our subsequent design, synthesis, and evaluation of proteolysis-targeting chimeric (PROTAC) small-molecule BET degraders. Our efforts have led to the discovery of extremely potent BET degraders, exemplified by QCA570, which effectively induces degradation of BET proteins and inhibits cell growth in human acute leukemia cell lines even at low picomolar concentrations. QCA570 achieves complete and durable tumor regression in leukemia xenograft models in mice at well-tolerated dose-schedules. QCA570 is the most potent and efficacious BET degrader reported to date.
Asunto(s)
Diseño de Fármacos , Proteínas/metabolismo , Proteolisis/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Ratones , Modelos Moleculares , Conformación Proteica , Proteínas/química , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacocinéticaRESUMEN
The structure-based design of M-525 as the first-in-class, highly potent, irreversible small-molecule inhibitor of the menin-MLL interaction is presented. M-525 targets cellular menin protein at sub-nanomolar concentrations and achieves low nanomolar potencies in cell growth inhibition and in the suppression of MLL-regulated gene expression in MLL leukemia cells. M-525 demonstrates high cellular specificity over non-MLL leukemia cells and is more than 30 times more potent than its corresponding reversible inhibitors. Mass spectrometric analysis and co-crystal structure of M-525 in complex with menin firmly establish its mode of action. A single administration of M-525 effectively suppresses MLL-regulated gene expression in tumor tissue. An efficient procedure was developed to synthesize M-525. This study demonstrates that irreversible inhibition of menin may be a promising therapeutic strategy for MLL leukemia.
Asunto(s)
Antineoplásicos/farmacología , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , Proteína de la Leucemia Mieloide-Linfoide/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Antineoplásicos/química , Sitios de Unión , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Diseño de Fármacos , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Simulación de Dinámica Molecular , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Dominios y Motivos de Interacción de Proteínas/efectos de los fármacos , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Relación Estructura-ActividadRESUMEN
The bromodomain and extra-terminal (BET) family proteins, consisting of BRD2, BRD3, BRD4, and testis-specific BRDT members, are epigenetic "readers" and play a key role in the regulation of gene transcription. BET proteins are considered to be attractive therapeutic targets for cancer and other human diseases. Recently, heterobifunctional small-molecule BET degraders have been designed based upon the proteolysis targeting chimera (PROTAC) concept to induce BET protein degradation. Herein, we present our design, synthesis, and evaluation of a new class of PROTAC BET degraders. One of the most promising compounds, 23, effectively degrades BRD4 protein at concentrations as low as 30 pM in the RS4;11 leukemia cell line, achieves an IC50 value of 51 pM in inhibition of RS4;11 cell growth and induces rapid tumor regression in vivo against RS4;11 xenograft tumors. These data establish that compound 23 (BETd-260/ZBC260) is a highly potent and efficacious BET degrader.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Indoles/química , Indoles/farmacología , Proteínas Nucleares/metabolismo , Pirimidinas/química , Pirimidinas/farmacología , Factores de Transcripción/metabolismo , Animales , Antineoplásicos/síntesis química , Proteínas de Ciclo Celular , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Diseño de Fármacos , Ensayos de Selección de Medicamentos Antitumorales/métodos , Femenino , Humanos , Indoles/síntesis química , Leucemia/tratamiento farmacológico , Ratones SCID , Proteínas Nucleares/química , Proteínas Serina-Treonina Quinasas/metabolismo , Proteolisis , Pirimidinas/síntesis química , Proteínas de Unión al ARN/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Relación Estructura-Actividad , Factores de Transcripción/química , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The Cullin-RING E3 ubiquitin ligases (CRLs) regulate homeostasis of ~20% of cellular proteins and their activation require neddylation of their cullin subunit. Cullin neddylation is modulated by a scaffolding DCN protein through interactions with both the cullin protein and an E2 enzyme such as UBC12. Here we report the development of DI-591 as a high-affinity, cell-permeable small-molecule inhibitor of the DCN1-UBC12 interaction. DI-591 binds to purified recombinant human DCN1 and DCN2 proteins with K i values of 10-12 nM, and disrupts the DCN1-UBC12 interaction in cells. Treatment with DI-591 selectively converts cellular cullin 3 into an un-neddylated inactive form with no or minimum effect on other cullin members. Our data firmly establish a previously unrecognized specific role of the DCN1-UBC12 interaction for cellular neddylation of cullin 3. DI-591 is an excellent probe compound to investigate the role of the cullin 3 CRL ligase in biological processes and human diseases.
Asunto(s)
Proteínas Cullin/metabolismo , Morfolinas/farmacología , Proteínas Proto-Oncogénicas/metabolismo , Tiazoles/farmacología , Enzimas Ubiquitina-Conjugadoras/metabolismo , Química Farmacéutica , Clonación Molecular , Biología Computacional , Cristalografía por Rayos X , Diseño de Fármacos , Células HeLa , Células Hep G2 , Humanos , Concentración 50 Inhibidora , Péptidos y Proteínas de Señalización Intracelular , Cinética , Permeabilidad , Unión Proteica , Dominios Proteicos , Proteínas , Proteínas Recombinantes/metabolismoRESUMEN
Triple-negative breast cancers (TNBC) remain clinically challenging with a lack of options for targeted therapy. In this study, we report the development of a second-generation BET protein degrader, BETd-246, which exhibits superior selectivity, potency, and antitumor activity. In human TNBC cells, BETd-246 induced degradation of BET proteins at low nanomolar concentrations within 1 hour of exposure, resulting in robust growth inhibition and apoptosis. BETd-246 was more potent and effective in TNBC cells than its parental BET inhibitor compound BETi-211. RNA-seq analysis revealed predominant downregulation of a large number of genes involved in proliferation and apoptosis in cells treated with BETd-246, as compared with BETi-211 treatment that upregulated and downregulated a similar number of genes. Functional investigations identified the MCL1 gene as a critical downstream effector for BET degraders, which synergized with small-molecule inhibitors of BCL-xL in triggering apoptosis. In multiple murine xenograft models of human breast cancer, BETd-246 and a further optimized analogue BETd-260 effectively depleted BET proteins in tumors and exhibited strong antitumor activities at well-tolerated dosing schedules. Overall, our findings show that targeting BET proteins for degradation represents an effective therapeutic strategy for TNBC treatment. Cancer Res; 77(9); 2476-87. ©2017 AACR.
Asunto(s)
Antineoplásicos/administración & dosificación , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteolisis/efectos de los fármacos , Receptores de Superficie Celular/antagonistas & inhibidores , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Animales , Línea Celular Tumoral , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Ratones , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Proteínas del Tejido Nervioso/genética , Receptores de Superficie Celular/genética , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Castration resistant prostate cancer (CRPC) is a deadly disease with few therapeutic options once patients become resistant to second generation drugs targeting the AR-transcriptional program. The BET-BRD readers of chromatin are key regulators of AR-, ERG-, and c-Myc-mediated transcription in CRPC. BET-BRD inhibitors have demonstrated pre-clinical efficacy in models of CRPC and are currently being evaluated in several clinical trials. These novel drugs have the potential to transform the way we treat CRPC in the near future.
Asunto(s)
Proteínas Nucleares/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Factores de Transcripción/metabolismo , Animales , Antineoplásicos/uso terapéutico , Humanos , Masculino , Proteínas Nucleares/antagonistas & inhibidores , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Receptores Androgénicos/metabolismo , Transducción de Señal , Factores de Transcripción/antagonistas & inhibidoresRESUMEN
Crizotinib is the first anaplastic lymphoma kinase (ALK) inhibitor to have been approved for the treatment of non-small cell lung cancer (NSCLC) harboring an ALK fusion gene, but it has been found that, in the clinic, patients develop resistance to it. Alectinib and ceritinib are second-generation ALK inhibitors which show remarkable clinical responses in both crizotinib-naive and crizotinib-resistant NSCLC patients harboring an ALK fusion gene. Despite their impressive activity, clinical resistance to alectinib and ceritinib has also emerged. In the current study, we elucidated the resistance mechanisms to these second-generation ALK inhibitors in the H3122 NSCLC cell line harboring the EML4-ALK variant 1 fusion in vitro. Prolonged treatment of the parental H3122 cells with alectinib and ceritinib led to two cell lines which are 10 times less sensitive to alectinib and ceritinib than the parental H3122 cell line. Although mutations of ALK in its kinase domain are a common resistance mechanism for crizotinib, we did not detect any ALK mutation in these resistant cell lines. Rather, overexpression of phospho-ALK and alternative receptor tyrosine kinases such as phospho-EGFR, phospho-HER3, and phospho-IGFR-1R was observed in both resistant cell lines. Additionally, NRG1, a ligand for HER3, is upregulated and responsible for resistance by activating the EGFR family pathways through the NRG1-HER3-EGFR axis. Combination treatment with EGFR inhibitors, in particular afatinib, was shown to be effective at overcoming resistance. Our study provides new mechanistic insights into adaptive resistance to second-generation ALK inhibitors and suggests a potential clinical strategy to combat resistance to these second-generation ALK inhibitors in NSCLC.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Resistencia a Antineoplásicos/genética , Receptores ErbB/biosíntesis , Neurregulina-1/biosíntesis , Proteínas Tirosina Quinasas Receptoras/genética , Quinasa de Linfoma Anaplásico , Carbazoles/administración & dosificación , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Resistencia a Antineoplásicos/efectos de los fármacos , Receptores ErbB/antagonistas & inhibidores , Regulación Neoplásica de la Expresión Génica , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial/biosíntesis , Humanos , Mutación , Neurregulina-1/genética , Proteínas de Fusión Oncogénica/genética , Piperidinas/administración & dosificación , Pirimidinas/administración & dosificación , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Receptor ErbB-3/biosíntesis , Transducción de Señal/efectos de los fármacos , Sulfonas/administración & dosificaciónRESUMEN
Botulinum neurotoxin type-A (BoNT/A), as onabotulinumtoxinA, is approved globally for 11 major therapeutic and cosmetic indications. While the mechanism of action for BoNT/A at the presynaptic nerve terminal has been established, questions remain regarding intracellular trafficking patterns and overall fate of the toxin. Resolving these questions partly depends on the ability to detect BoNT/A's location, distribution, and movement within a cell. Due to BoNT/A's high potency and extremely low concentrations within neurons, an alternative approach has been employed. This involves utilizing specific antibodies against the BoNT/A-cleaved SNAP25 substrate (SNAP25197) to track the enzymatic activity of toxin within cells. Using our highly specific mouse monoclonal antibody (mAb) against SNAP25197, we generated human and murine recombinant versions (rMAb) using specific backbone immunoglobulins. In this study, we validated the specificity of our anti-SNAP25197 rMAbs in several different assays and performed side-by-side comparisons to commercially-available and in-house antibodies against SNAP25. Our rMAbs were highly specific for SNAP25197 in all assays and on several different BoNT/A-treated tissues, showing no cross-reactivity with full-length SNAP25. This was not the case with other reportedly SNAP25197-selective antibodies, which were selective in some, but not all assays. The rMAbs described herein represent effective new tools for detecting BoNT/A activity within cells.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos/inmunología , Toxinas Botulínicas Tipo A/inmunología , Proteína 25 Asociada a Sinaptosomas/antagonistas & inhibidores , Proteína 25 Asociada a Sinaptosomas/inmunología , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/farmacología , Western Blotting , Toxinas Botulínicas Tipo A/metabolismo , Toxinas Botulínicas Tipo A/farmacología , Células Cultivadas , Humanos , Inmunohistoquímica , Masculino , Ratones , Microscopía Confocal , Neuronas/efectos de los fármacos , Neuronas/inmunología , Neuronas/metabolismo , Fragmentos de Péptidos/inmunología , Transporte de Proteínas , Ratas Sprague-Dawley , Proteínas Recombinantes , Piel/efectos de los fármacos , Piel/inmunología , Piel/metabolismo , Especificidad por Sustrato , Vejiga Urinaria/efectos de los fármacos , Vejiga Urinaria/inmunología , Vejiga Urinaria/metabolismoRESUMEN
Septins are a family of 14 cytoskeletal proteins that dynamically form hetero-oligomers and organize membrane microdomains for protein complexes. The previously reported interactions with SNARE proteins suggested the involvement of septins in exocytosis. However, the contradictory results of up- or down-regulation of septin-5 in various cells and mouse models or septin-4 in mice suggested either an inhibitory or a stimulatory role for these septins in exocytosis. The involvement of the ubiquitously expressed septin-2 or general septin polymerization in exocytosis has not been explored to date. Here, by nano-LC with tandem MS and immunoblot analyses of the septin-2 interactome in mouse brain, we identified not only SNARE proteins but also Munc-18-1 (stabilizes assembled SNARE complexes), N-ethylmaleimide-sensitive factor (NSF) (disassembles SNARE complexes after each membrane fusion event), and the chaperones Hsc70 and synucleins (maintain functional conformation of SNARE proteins after complex disassembly). Importantly, α-soluble NSF attachment protein (SNAP), the adaptor protein that mediates NSF binding to the SNARE complex, did not interact with septin-2, indicating that septins undergo reorganization during each exocytosis cycle. Partial depletion of septin-2 by siRNA or impairment of septin dynamics by forchlorfenuron inhibited constitutive and stimulated exocytosis of secreted and transmembrane proteins in various cell types. Forchlorfenuron impaired the interaction between SNAP-25 and its chaperone Hsc70, decreasing SNAP-25 levels in cultured neuroendocrine cells, and inhibited both spontaneous and stimulated acetylcholine secretion in mouse motor neurons. The results demonstrate a stimulatory role of septin-2 and the dynamic reorganization of septin oligomers in exocytosis.
Asunto(s)
Encéfalo/metabolismo , Exocitosis , Proteoma/metabolismo , Septinas/metabolismo , Animales , Western Blotting , Encéfalo/efectos de los fármacos , Línea Celular , Línea Celular Tumoral , Perros , Femenino , Células HEK293 , Humanos , Células de Riñón Canino Madin Darby , Masculino , Ratones Endogámicos BALB C , Microscopía Confocal , Células PC12 , Compuestos de Fenilurea/farmacología , Unión Proteica/efectos de los fármacos , Multimerización de Proteína , Proteómica , Piridinas/farmacología , Interferencia de ARN , Ratas , Septinas/química , Septinas/genética , Proteína 25 Asociada a Sinaptosomas/metabolismoRESUMEN
Proteolytic cleavage of synaptosomal-associated protein 25 by the light chain of botulinum neurotoxin type A (LCA) results in a blockade of neurotransmitter release that persists for several months in motor neurons. The L428A/L429A mutation in LCA is known to significantly shorten both the proteolytic and neuroparalytic effects of the neurotoxin in mice. To elucidate the cellular mechanism for LCA longevity, we studied the effects of L428A/L429A mutation on the interactome, localization and stability of LCA expressed in cultured neuronal cells. Mass spectrometry analysis of the LCA interactome showed that the mutation prevented the interaction of LCA with septins. The wild-type LCA was concentrated in plasma-membrane-associated clusters, colocalizing with septins-2 and septin-7, which accumulated in these clusters only in the presence of LCA. The L428A/L429A mutation decreased co-clustering of LCA and septins and accelerated proteasomal and non-proteasomal degradation of LCA. Similarly, the impairment of septin oligomerization by forchlorfenuron or silencing of septin-2 prevented LCA interaction and clustering with septins and increased LCA degradation. Therefore, the dileucine-mediated LCA-septin co-clustering is crucial for the long-lasting stabilization of LCA-related proteolytic and presumably neuroparalytic activity.
Asunto(s)
Toxinas Botulínicas Tipo A/metabolismo , Membrana Celular/metabolismo , Neuronas/fisiología , Síndromes de Neurotoxicidad/metabolismo , Septinas/metabolismo , Animales , Toxinas Botulínicas Tipo A/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Humanos , Ratones , Mutación/genética , Neuronas/microbiología , Síndromes de Neurotoxicidad/microbiología , Compuestos de Fenilurea/farmacología , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Multimerización de Proteína/efectos de los fármacos , Estabilidad Proteica/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/genética , Piridinas/farmacología , ARN Interferente Pequeño/genética , Septinas/genéticaRESUMEN
Botulinum neurotoxin serotype A (BoNT/A) causes transient muscle paralysis by entering motor nerve terminals (MNTs) where it cleaves the SNARE protein Synaptosomal-associated protein 25 (SNAP25206) to yield SNAP25197. Cleavage of SNAP25 results in blockage of synaptic vesicle fusion and inhibition of the release of acetylcholine. The specific uptake of BoNT/A into pre-synaptic nerve terminals is a tightly controlled multistep process, involving a combination of high and low affinity receptors. Interestingly, the C-terminal binding domain region of BoNT/A, HC/A, is homologous to fibroblast growth factors (FGFs), making it a possible ligand for Fibroblast Growth Factor Receptors (FGFRs). Here we present data supporting the identification of Fibroblast Growth Factor Receptor 3 (FGFR3) as a high affinity receptor for BoNT/A in neuronal cells. HC/A binds with high affinity to the two extra-cellular loops of FGFR3 and acts similar to an agonist ligand for FGFR3, resulting in phosphorylation of the receptor. Native ligands for FGFR3; FGF1, FGF2, and FGF9 compete for binding to FGFR3 and block BoNT/A cellular uptake. These findings show that FGFR3 plays a pivotal role in the specific uptake of BoNT/A across the cell membrane being part of a larger receptor complex involving ganglioside- and protein-protein interactions.
Asunto(s)
Toxinas Botulínicas Tipo A/metabolismo , Membrana Celular/metabolismo , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/metabolismo , Animales , Toxinas Botulínicas Tipo A/genética , Membrana Celular/genética , Células HEK293 , Humanos , Ratones , Células PC12 , Transporte de Proteínas/genética , Ratas , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/genética , Proteína 25 Asociada a Sinaptosomas/genética , Proteína 25 Asociada a Sinaptosomas/metabolismoRESUMEN
Botulinum neurotoxin serotype A (BoNT/A), a potent therapeutic used to treat various disorders, inhibits vesicular neurotransmitter exocytosis by cleaving SNAP25. Development of cell-based potency assays (CBPAs) to assess the biological function of BoNT/A have been challenging because of its potency. CBPAs can evaluate the key steps of BoNT action: receptor binding, internalization-translocation, and catalytic activity; and therefore could replace the current mouse bioassay. Primary neurons possess appropriate sensitivity to develop potential replacement assays but those potency assays are difficult to perform and validate. This report describes a CBPA utilizing differentiated human neuroblastoma SiMa cells and a sandwich ELISA that measures BoNT/A-dependent intracellular increase of cleaved SNAP25. Assay sensitivity is similar to the mouse bioassay and measures neurotoxin biological activity in bulk drug substance and BOTOX® product (onabotulinumtoxinA). Validation of a version of this CBPA in a Quality Control laboratory has led to FDA, Health Canada, and European Union approval for potency testing of BOTOX®, BOTOX® Cosmetic, and Vistabel®. Moreover, we also developed and optimized a BoNT/A CBPA screening assay that can be used for the discovery of novel BoNT/A inhibitors to treat human disease.