RESUMO
3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase is the rate-limiting enzyme in the cholesterol biosynthetic pathway, and competitive inhibitors targeting the catalytic domain of this enzyme, so-called statins, are widely used for the treatment of hyperlipidemia. The membrane domain mediates the sterol-accelerated degradation, a post-translational negative feedback mechanism, and small molecules triggering such degradation have been studied as an alternative therapeutic option. Such strategies are expected to provide benefits over catalytic site inhibitors, as the inhibition leads to transcriptional and post-translational upregulation of the enzyme, necessitating a higher dose of the inhibitors and concomitantly increasing the risk of serious adverse effects, including myopathies. Through our previous study on SR12813, a synthetic small molecule that induces degradation of HMG-CoA reductase, we identified a nitrogen-containing bisphosphonate ester SRP3042 as a highly potent HMG-CoA reductase degrader. Here, we performed a systematic structure-activity relationship study to optimize its activity and physicochemical properties, specifically focusing on the reduction of lipophilicity. Mono-fluorination of tert-butyl groups on the molecules was found to increase the HMG-CoA reductase degradation activity while reducing lipophilicity, suggesting the mono-fluorination of saturated alkyl groups as a useful strategy to balance potency and lipophilicity of the lead compounds.
Assuntos
Inibidores de Hidroximetilglutaril-CoA Redutases , Oxirredutases , Animais , Cricetinae , Hidroximetilglutaril-CoA Redutases/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Colesterol/metabolismo , Células CHORESUMO
Cholesterol biosynthesis is a high-cost process and, therefore, tightly regulated by both transcriptional and posttranslational negative feedback mechanisms in response to the level of cellular cholesterol. Squalene monooxygenase (SM, also known as squalene epoxidase or SQLE) is a rate-limiting enzyme in the cholesterol biosynthetic pathway and catalyzes epoxidation of squalene. The stability of SM is negatively regulated by cholesterol via its N-terminal regulatory domain (SM-N100). In this study, using a SM-luciferase fusion reporter cell line, we performed a chemical genetics screen that identified inhibitors of SM itself as up-regulators of SM. This effect was mediated through the SM-N100 region, competed with cholesterol-accelerated degradation, and required the E3 ubiquitin ligase MARCH6. However, up-regulation was not observed with statins, well-established cholesterol biosynthesis inhibitors, and this pointed to the presence of another mechanism other than reduced cholesterol synthesis. Further analyses revealed that squalene accumulation upon treatment with the SM inhibitor was responsible for the up-regulatory effect. Using photoaffinity labeling, we demonstrated that squalene directly bound to the N100 region, thereby reducing interaction with and ubiquitination by MARCH6. Our findings suggest that SM senses squalene via its N100 domain to increase its metabolic capacity, highlighting squalene as a feedforward factor for the cholesterol biosynthetic pathway.
Assuntos
Esqualeno Mono-Oxigenase/metabolismo , Esqualeno/metabolismo , Regulação Alostérica , Benzilaminas , Colesterol/biossíntese , Retículo Endoplasmático/enzimologia , Células HEK293 , Humanos , Proteínas de Membrana/metabolismo , Proteostase , Esqualeno Mono-Oxigenase/antagonistas & inibidores , Tiofenos , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
HMG-CoA reductase (HMGCR) is the rate-limiting enzyme in the cholesterol biosynthetic pathway, and is the target of cholesterol-lowering drugs, statins. Previous studies have demonstrated that the enzyme activity is regulated by sterol-induced degradation in addition to transcriptional regulation through sterol-regulatory-element-binding proteins (SREBPs). While 25-hydroxycholesterol induces both HMGCR degradation and SREBP inhibition in a nonselective manner, lanosterol selectively induces HMGCR degradation. Here, to clarify the structural determinants of selectivity for the two activities, we established a luciferase-based assay monitoring HMGCR degradation and used it to profile the structure-activity/selectivity relationships of oxysterols and (oxy)lanosterols. We identified several sterols that selectively induce HMGCR degradation and one sterol, 25-hydroxycholest-4-en-3-one, that selectively inhibits the SREBP pathway. These results should be helpful in designing more potent and selective HMGCR degraders.
Assuntos
Hidroximetilglutaril-CoA Redutases/metabolismo , Lanosterol/metabolismo , Oxisteróis/metabolismo , Células HEK293 , Humanos , Lanosterol/farmacologia , Estrutura Molecular , Oxisteróis/farmacologia , Proteínas de Ligação a Elemento Regulador de Esterol/antagonistas & inibidores , Proteínas de Ligação a Elemento Regulador de Esterol/metabolismo , Relação Estrutura-AtividadeRESUMO
HMG-CoA reductase (HMGCR) is a rate-limiting enzyme in the cholesterol biosynthetic pathway, and its catalytic domain is the well-known target of cholesterol-lowering drugs, statins. HMGCR is subject to layers of negative feedback loops; excess cholesterol inhibits transcription of the gene, and lanosterols and oxysterols accelerate degradation of HMGCR. A class of synthetic small molecules, bisphosphonate esters exemplified by SR12813, has been known to induce accelerated degradation of HMGCR and reduce the serum cholesterol level. Although genetic and biochemical studies revealed that the accelerated degradation requires the membrane domain of HMGCR and Insig, an oxysterol sensor on the endoplasmic reticulum membrane, the direct target of the bisphosphonate esters remains unclear. In this study, we developed a potent photoaffinity probe of the bisphosphonate esters through preliminary structure-activity relationship study and demonstrated binding of the bisphosphonate esters to the HMGCR membrane domain. These results provide an important clue to understand the elusive mechanism of the SR12813-mediated HMGCR degradation and serve as a basis to develop more potent HMGCR degraders that target the non-catalytic, membrane domain of the enzyme.
Assuntos
Difosfonatos/farmacologia , Hidroximetilglutaril-CoA Redutases/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Células Cultivadas , Difosfonatos/síntese química , Difosfonatos/química , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/síntese química , Inibidores de Hidroximetilglutaril-CoA Redutases/química , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Selective estrogen receptor (ER) down-regulators (SERDs) are pure ER antagonists that also induce ER degradation upon binding to the receptor. Although SERDs have been developed for the treatment of ER-positive breast cancers for nearly a decade, their precise mechanism(s) of action and structure-activity relationship are still unclear. Generally, Western blotting is used to examine the effects of SERDs on ER protein levels, but the methodology is low-throughput and not quantitative. Here, we describe a quantitative, high-throughput, luciferase-based assay for the evaluation of SERDs activity. For this purpose, we established stable recombinant HEK-293 cell lines expressing ERα fused with emerald luciferase. We also designed and synthesized new diphenylmethane derivatives as candidate SERDs, and evaluated their SERDs activity using the developed system in order to examine their structure-activity relationship, taking EC50 as a measure of potency, and Emax as a measure of efficacy.
Assuntos
Compostos Benzidrílicos/química , Regulação para Baixo/efeitos dos fármacos , Receptor alfa de Estrogênio/antagonistas & inibidores , Compostos Benzidrílicos/farmacologia , Sítios de Ligação , Ciclofenil/química , Ciclofenil/metabolismo , Antagonistas de Estrogênios/química , Antagonistas de Estrogênios/metabolismo , Antagonistas de Estrogênios/farmacologia , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Células HEK293 , Humanos , Simulação de Acoplamento Molecular , Fenóis/química , Fenóis/farmacologia , Ligação Proteica , Estrutura Terciária de Proteína , Relação Estrutura-AtividadeRESUMO
N-Benzyl-N-(4-phenoxyphenyl)benzenesulfonamide derivatives were developed as a novel class of nonsteroidal glucocorticoid receptor (GR) modulators, which are promising drug candidates for treating immune-related disorders. Focusing on the similarity of the GR and progesterone receptor (PR) ligand-binding domain (LBD) structures, we adopted our recently developed PR antagonist 10 as a lead compound and synthesized a series of derivatives. We found that the N-(4-phenoxyphenyl)benzenesulfonamide skeleton serves as a versatile scaffold for GR antagonists. Among them, 4-cyano derivative 14m was the most potent, with an IC50 value of 1.43µM for GR. This compound showed good selectivity for GR; it retained relatively weak antagonistic activity toward PR (IC50 for PR: 8.00µM; 250-fold less potent than 10), but showed no activity toward AR, ERα or ERß. Interestingly, the 4-amino derivative 15a exhibited transrepression activity toward NF-κB in addition to GR-antagonistic activity, whereas 14m did not. The structure-activity relationship for transrepression was different from that for GR-antagonistic activity. Computational docking simulations suggested that 15a might bind to the ligand-binding pocket of GR in a different manner from 14m. These findings open up new possibilities for developing novel nonsteroidal GR modulators with distinctive activity profiles.
Assuntos
Receptores de Glucocorticoides/antagonistas & inibidores , Sulfonamidas/farmacologia , Relação Dose-Resposta a Droga , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Receptores de Glucocorticoides/metabolismo , Relação Estrutura-Atividade , Sulfonamidas/síntese química , Sulfonamidas/químicaRESUMO
BACKGROUND: The preoperative imaging diagnosis of the distal portion of the internal carotid artery (ICA) is extremely important for carotid endarterectomy (CEA). Herein the authors defined a line from the C1 transverse process to the hyoid bone (C1-H line) and evaluated whether the line can be used to predict an accessible ICA in CEA. METHODS: A cross point between the C1-H line and distal ICA was analyzed using three-dimensional computerized tomographic angiography (3D-CTA) in 20 patients. The C1-H line was compared to the line drawn from the mastoid process to the mandible (M-M line). Intraoperative exposure of the distal ICA was evaluated using both lines. Furthermore, the distance of each line from the C2 vertebra was measured to identify the distance difference of each line in relation to the cervical posture. RESULTS: A distal ICA exposed at a cross point of the C1-H line corresponded well with the intraoperative findings. The cross point between the C1-H line and distal ICA was positioned at an average of 7.0 ± 0.7 mm cranially in comparison to the M-M line. The C1-H line showed smaller distance differences at different cervical positions than the M-M line. The C1-H line moved an average of 2.8 ± 2.5 mm from a cervical neutral position to an extensional one in the perpendicular direction. CONCLUSION: The C1-H line measured by 3D-CTA is a simple and useful indicator of the distal ICA exposure in the preoperative diagnosis for CEA.
Assuntos
Artéria Carótida Interna/diagnóstico por imagem , Angiografia Cerebral/métodos , Endarterectomia das Carótidas/métodos , Osso Hioide/diagnóstico por imagem , Adulto , Idoso , Artéria Carótida Interna/cirurgia , Feminino , Humanos , Osso Hioide/cirurgia , Masculino , Pessoa de Meia-Idade , Coluna Vertebral/diagnóstico por imagemRESUMO
BACKGROUND: We verified the effectiveness of training in endoscopic endonasal transsphenoidal surgery (eETSS) techniques using chicken eggs and a skull model. METHODS: We verified the area of eggshell removed by drilling when five residents and four experts used the chicken eggs and a skull model. RESULTS: When residents performed drilling on 10 eggs, a mean (± standard deviation [SD]) area of 31.2 ± 17.5 mm2 was removed from the first egg, and 104.8 ± 3.3 mm2 from the tenth and final egg, representing an increase in area and a decrease in SD. The experts performed the same drilling operation on a single egg, and removed a mean area of 257± 31.7 mm2. These results demonstrated that skills improved as a result of this training, and suggested that this method was also capable of overcoming the initial individual differences in the amount of force applied and ability. An obvious difference between residents and experts was seen in the area removed (p = 0.00011); however, this was attributed to differences in endoscopic manipulation, rather than drilling skill. CONCLUSION: Our findings suggest that this training method could be adequate for acquiring eETSS techniques. Although experts showed superior endoscopic manipulation, residents may also be able to acquire adequate endoscopic skills through further training, and our training method appears to offer an effective means of improving eETSS techniques.
Assuntos
Competência Clínica , Ovos , Endoscopia/métodos , Cavidade Nasal/cirurgia , Neurocirurgia/educação , Procedimentos Neurocirúrgicos/métodos , Crânio/cirurgia , Osso Esfenoide/cirurgia , Animais , Galinhas , Casca de Ovo , Humanos , Internato e ResidênciaRESUMO
BACKGROUND/AIM: Resection of brain metastases is a well-established treatment modality that can prolong the survival of patients for whom surgery is indicated. Whole-brain radiotherapy (WBRT) has been the standard postoperative therapy. In recent years, however, clinicians have increasingly avoided WBRT due to its associated adverse events. This study investigated the impact of postoperative WBRT and systemic chemotherapy as prognostic factors on the survival of patients who had undergone resection of brain metastases. PATIENTS AND METHODS: The study subjects were 172 patients who underwent surgical resection for brain metastases. Comparative analyses of survival after WBRT and systemic chemotherapy were performed. RESULTS: Postoperative WBRT had no survival-prolonging effect, whereas postoperative systemic chemotherapy prolonged survival. A comparison based on the number of systemic chemotherapy regimens administered prior to surgery showed that fewer regimens correlated with a better prognosis. CONCLUSION: The addition of WBRT after surgical resection of brain metastases is no longer a standard treatment strategy and systemic chemotherapy after surgery is a positive prognostic factor.
Assuntos
Neoplasias Encefálicas , Humanos , Neoplasias Encefálicas/secundário , Neoplasias Encefálicas/terapia , Neoplasias Encefálicas/cirurgia , Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/radioterapia , Masculino , Feminino , Pessoa de Meia-Idade , Idoso , Adulto , Prognóstico , Irradiação Craniana/métodos , Terapia Combinada , Idoso de 80 Anos ou maisRESUMO
Cells are covered with a thick layer of sugar molecules known as glycans. Abnormal glycosylation is a hallmark of cancer, and hypersialylation increases tumor metastasis by promoting immune evasion and inducing tumor cell invasion and migration. Inhibiting sialylation is thus a potential anticancer treatment strategy. However, targeting sialic acids is difficult because of the lack of selective delivery tools. Here, we present a prodrug strategy for selectively releasing the global inhibitor of sialylation peracetylated 3Fax-Neu5Ac (PFN) in cancer cells using the reaction between phenyl azide and endogenous acrolein, which is overproduced in most cancer cells. The prodrug significantly suppressed tumor growth in mice as effectively as PFN without causing kidney dysfunction, which is associated with PFN. The use of sialylated glycans as immune checkpoints is gaining increasing attention, and the proposed method for precisely targeting aberrant sialylation provides a novel avenue for expanding current cancer treatments.
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The direct synthesis of drugs in vivo enables drugs to treat diseases without causing side effects in healthy tissues. Transition-metal reactions have been widely explored for uncaging and synthesizing bioactive drugs in biological environments because of their remarkable reactivity. Nonetheless, it is difficult to develop a promising method to achieve in vivo drug synthesis because blood cells and metabolites deactivate transition-metal catalysts. We report that a robust albumin-based artificial metalloenzyme (ArM) with a low loading (1-5 mol%) can promote Ru-based olefin metathesis to synthesize molecular scaffolds and an antitumor drug in blood. The ArM retained its activity after soaking in blood for 24 h and provided the first example of catalytic olefin cross metathesis in blood. Furthermore, the cyclic-Arg-Gly-Asp (cRGD) peptide-functionalized ArM at lower dosages could still efficiently perform in vivo drug synthesis to inhibit the growth of implanted tumors in mice. Such a system can potentially construct therapeutic drugs in vivo for therapies without side effects.
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BACKGROUND/AIM: Standard treatment options for primary central nervous system lymphoma (PCNSL) include high-dose methotrexate (HD-MTX)-based drug therapy and whole-brain radiation therapy. However, there are many cases in which these standard treatment options are not tolerated for various reasons. In the present study, five cases of refractory/relapsed PCNSL that are difficult to treat with standard treatment were successfully treated by tirabrutinib. PATIENTS AND METHODS: A total of 5 patients (4 women, 1 man) with refractory (n=3) and relapsed (n=2) PCNSL were included. The patients had a median age of 76 years and a median Karnofsky performance status (KPS) of 40. The reasons why standard treatment cannot be given to these patients are the low KPS, renal dysfunction, and resistance to HD-MTX. Administration of a drug via the oral route was challenging in three patients; thus, these patients were administered tirabrutinib in suspension through a nasogastric tube. RESULTS: Imaging findings showed that the patients achieved a 100% response rate to tirabrutinib, with a median survival of 8 months. As symptoms improved, 2 of the 3 patients who were initially administered tirabrutinib via a nasogastric tube were able to receive the drug via the oral route. Three patients developed adverse reactions; however, treatment was not interrupted because they were manageable. CONCLUSION: Tirabrutinib was effective in the treatment of patients who were unable to receive standard treatment options. Tirabrutinib may be considered one of the novel treatment strategies that could improve the prognosis of PCNSL patients in the future.
Assuntos
Neoplasias Encefálicas , Neoplasias do Sistema Nervoso Central , Linfoma não Hodgkin , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Sistema Nervoso Central , Irradiação Craniana , Feminino , Humanos , Imidazóis , Linfoma não Hodgkin/tratamento farmacológico , Masculino , Metotrexato , Pirimidinas , Estudos RetrospectivosRESUMO
Objective: Transvenous embolization (TVE) is typically used in combination with the residual shunt of transarterial embolization (TAE) for the treatment of direct carotid-cavernous fistulas (direct CCFs). This report is about our additional embolization method using combination therapy. Case Presentation: Five consecutive cases of direct CCF were presented; two were caused by aneurysms and three by head injuries. The treatment for each was started with TAE, with the addition of TVE if a shunt remained. At the time of TVE, a microcatheter positioned in the internal carotid artery passing from the cavernous sinus through the aneurysm neck or fistula was pulled back (pull-back method). It was then placed in the coil mass with TAE, and additional coils were filled. In two cases, the shunt disappeared by using only TAE, whereas it disappeared after being additionally embolized by the pull-back method in the remaining cases. All patients recovered with no postoperative complications. Conclusion: The TAE and TVE combination therapy with the pull-back method could efficiently embolize the residual shunt after TAE.
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Glutathione peroxidase 4 (GPX4) is an intracellular enzyme that oxidizes glutathione while reducing lipid peroxides and is a promising target for cancer therapy. To date, several GPX4 inhibitors have been reported to exhibit cytotoxicity against cancer cells. However, some cancer cells are less sensitive to the known GPX4 inhibitors. This study aimed to explore compounds showing synergistic effects with GPX4 inhibitors. We screened a chemical library and identified a compound named NPD4928, whose cytotoxicity was enhanced in the presence of a GPX4 inhibitor. Furthermore, we identified ferroptosis suppressor protein 1 as its target protein. The results indicate that NPD4928 enhanced the sensitivity of various cancer cells to GPX4 inhibitors, suggesting that the combination might have therapeutic potential via the induction of ferroptosis.
Assuntos
Ferroptose , Glutationa/metabolismo , Oxirredução , Fosfolipídeo Hidroperóxido Glutationa Peroxidase , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
BACKGROUND/AIM: Leptomeningeal carcinomatosis (LMC) with hydrocephalus is particularly difficult to treat, and its prognosis is extremely poor. The therapeutic outcomes of 14 patients with LMC-associated hydrocephalus who were treated with cerebrospinal fluid shunting are reported. PATIENTS AND METHODS: The study subjects were 14 LMC patients with solid primary cancer who had developed hydrocephalus. RESULTS: Postoperatively, both symptoms and Karnofsky performance status improved in 100% of patients. Postoperative therapy consisted of whole-brain radiotherapy in 4 cases and molecular targeted therapy in 4, with 6 patients not receiving any postoperative treatment. Median overall survival was 3.7 months, with no significant difference between those who underwent postoperative therapy and those who did not. However, two of those who received molecular targeted therapy survived for more than one year. CONCLUSION: Cerebrospinal fluid shunting for LMC-associated hydrocephalus is an effective therapeutic procedure from the palliative viewpoint. Patients for whom molecular targeted therapy is indicated may have better long-term survival.
Assuntos
Derivações do Líquido Cefalorraquidiano , Hidrocefalia/cirurgia , Carcinomatose Meníngea/cirurgia , Cuidados Paliativos , Adulto , Idoso , Feminino , Humanos , Hidrocefalia/etiologia , Hidrocefalia/mortalidade , Hidrocefalia/terapia , Estimativa de Kaplan-Meier , Masculino , Carcinomatose Meníngea/complicações , Carcinomatose Meníngea/mortalidade , Carcinomatose Meníngea/terapia , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Cavidade PeritonealRESUMO
We report that tirabrutinib was administered via nasogastric tubes to treat an elderly patient with primary central nervous system lymphoma (PCNSL). The patient was a 76-year-old woman who underwent endoscopic biopsy of multiple intracerebral masses, which resulted in the diagnosis of diffuse large B-cell lymphoma. The patient was diagnosed with PCNSL and was started on an induction regimen of systemic chemotherapy with rituximab in combination with high-dose methotrexate. However, after the second cycle of chemotherapy, the tumor grew rapidly, and the patient went into a coma. As a result, the treatment was changed to nasogastric tube administration of tirabrutinib suspension. After 1 week of tirabrutinib administration, the patient's level of consciousness improved, and furthermore, after 2 weeks of tirabrutinib administration, the patient was able to take tirabrutinib orally. Although oral administration is the standard route of administration for tirabrutinib, this case study showed that the nasogastric tube administration of tirabrutinib suspension is a therapeutic option for patients with impaired consciousness or dysphagia.
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BACKGROUND/AIM: Recent advances in systemic chemotherapy, including molecularly targeted therapy, have dramatically improved survival for patients with advanced non-small cell lung cancer. We retrospectively analyzed the clinical outcomes of surgical resection for brain metastases of non-small cell lung cancer cases performed at the Department of Neurosurgery of Kindai University Hospital, Osaka, Japan. PATIENTS AND METHODS: Craniotomy and tumor resection were performed for 56 patients with brain metastases of non-small cell lung cancer. Adenocarcinoma was the most common histological type, appearing in 40 cases, of which 18 were positive for driver gene mutations. RESULTS: Median survival for all 56 patients was 14.5 months, and single brain metastasis and adenocarcinoma were identified as favorable prognostic factors. Analysis limited to the 40 cases of adenocarcinoma identified single brain metastasis as a favorable prognostic factor. Although no significant difference was found for systemic chemotherapy, patients who received molecularly targeted therapy showed a better prognosis than those who received cytotoxic chemotherapy. Analyses of both the entire group and of adenocarcinoma patients alone found that whole-brain radiotherapy showed no significant association with survival. CONCLUSION: Single brain metastasis and adenocarcinoma were identified as favorable prognostic factors, but did not confirm any benefit from whole-brain radiotherapy. These results suggest that multimodal treatment strategies utilizing various methods of treatment, including systemic chemotherapy, may help prolong patient survival in the future.
Assuntos
Neoplasias Encefálicas/cirurgia , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/patologia , Idoso , Idoso de 80 Anos ou mais , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/secundário , Feminino , Humanos , Japão , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Retrospectivos , Taxa de SobrevidaRESUMO
Niemann-Pick disease type C is a rare, fatal neurodegenerative disorder characterized by massive intracellular accumulation of cholesterol. In most cases, loss-of-function mutations in the NPC1 gene that encodes lysosomal cholesterol transporter NPC1 are responsible for the disease, and more than half of the mutations are considered to interfere with the biogenesis or folding of the protein. We previously identified a series of oxysterol derivatives and phenanthridine-6-one derivatives as pharmacological chaperones, i.e., small molecules that can rescue folding-defective phenotypes of mutated NPC1, opening up an avenue to develop chaperone therapy for Niemann-Pick disease type C. Here, we present an improved image-based screen for NPC1 chaperones and we describe its application for drug-repurposing screening. We identified some azole antifungals, including itraconazole and posaconazole, and a kinase inhibitor, lapatinib, as probable pharmacological chaperones. A photo-crosslinking study confirmed direct binding of itraconazole to a representative folding-defective mutant protein, NPC1-I1061T. Competitive photo-crosslinking experiments suggested that oxysterol-based chaperones and itraconazole share the same or adjacent binding site(s), and the sensitivity of the crosslinking to P691S mutation in the sterol-sensing domain supports the hypothesis that their binding sites are located near this domain. Although the azoles were less effective in reducing cholesterol accumulation than the oxysterol-derived chaperones or an HDAC inhibitor, LBH-589, our findings should offer new starting points for medicinal chemistry efforts to develop better pharmacological chaperones for NPC1.
Assuntos
Descoberta de Drogas/métodos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Doença de Niemann-Pick Tipo C/tratamento farmacológico , Dobramento de Proteína/efeitos dos fármacos , Reposicionamento de Medicamentos/métodos , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/análise , Mutação/efeitos dos fármacos , Proteína C1 de Niemann-Pick , Doença de Niemann-Pick Tipo C/genética , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
Luciferase-based reporter assays are one of the most common cell-based screening formats for drug discovery, and simultaneous evaluation of the cytotoxic effect of test compounds is of great value in reducing false-positives. Here we share a multiplex assay protocol that allows sequential measurement of cell viability (cell number) and luciferase activity of the same sample in a multi-well-plate format. The viability assay employs a fluorogenic esterase substrate, CytoRed. â¢This protocol allows sequential measurement of endogenous esterase activity (as a surrogate for cell number) and then luciferase activity in a single sample.â¢The protocol eliminates the need for parallel viability assay or protein assay using separate aliquots of the lysate.â¢This protocol is especially useful for assays with cells stably expressing a luciferase construct, for which co-transfection of another reporter gene is not a viable option.
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BACKGROUND: Glioblastoma multiforme (GBM) is a malignant brain tumor with an extremely poor prognosis. GBM tissues frequently express mesenchymal-epithelial transition factor (MET), which induces cell division, growth and migration. In addition, angiogenesis is a significant feature of GBM, attributable to the overexpression of vascular endothelial growth factor (VEGF). Although the VEGF inhibitor bevacizumab was recently highlighted as the second-line drug for GBM treatment, GBMs often recur even with bevacizumab therapy. Based on these findings, we hypothesized that inhibition of both MET and VEGF would exhibit a synergistic effect on MET-overexpressing GBM. MATERIALS AND METHODS: As we observed MET expression at high levels in some patients with GBM, we designed GL261 murine glioma-based experiments. GL261 cells were transfected with siRNAs specific for MET and VEGF in vitro, and the cell growth ratios were evaluated. Simultaneously, transfected GL261 cells were transplanted into the brain of C57BL/6 mice, and their survival was monitored. RESULTS: GBM tissues frequently overexpressed MET protein at high levels compared with lower-grade gliomas. These GBMs at first responded to bevacizumab, but often eventually recurred. When GL261 cells were co-transfected with both MET-specific siRNA and VEGF-specific siRNA, the in vitro tumor cell growth significantly decelerated compared to single siRNA transfection. Consistently, when mice were transplanted with co-transfected GL261 cells, their survival was significantly prolonged compared to those given cells transfected with single siRNA. CONCLUSION: The current data indicate that the inhibition of both MET and VEGF exhibits efficient therapeutic effects of GBM-bearing hosts.