RESUMO
BACKGROUND AIMS: Several anti-mesothelin (MSLN) chimeric antigen receptor (CAR) T cells are in phase 1/2 clinical trials to treat solid-organ malignancies. The effect of MSLN antigen density on MSLN CAR cytotoxicity against tumor cells has not been examined previously, nor are there data regarding the effect of agents that increase MSLN antigen density on anti-MSLN CAR T cell efficacy. METHODS: MSLN antigen density was measured on a panel of pancreatic cancer and mesothelioma cell lines by flow cytometry. In parallel, the cytotoxicity and specificity of two anti-MSLN CAR T cells (m912 and SS1) were compared against these cell lines using a real-time impedance-based assay. The effect of two MSLN 'sheddase' inhibitors (lanabecestat and TMI-1) that increase MSLN surface expression was also tested in combination with CAR T cells. RESULTS: SS1 CAR T cells were more cytotoxic compared with m912 CAR T cells against cell lines that expressed fewer than â¼170 000 MSLN molecules/cell. A comparison of the m912 and amatuximab (humanized SS1) antibodies identified that amatuximab could detect and bind to lower levels of MSLN on pancreatic cancer and mesothelioma cell lines, suggesting that superior antibody/scFv affinity was the reason for the SS1 CAR's superior cytotoxicity. The cytotoxicity of m912 CAR T cells was improved in the presence of sheddase inhibitors, which increased MSLN antigen density. CONCLUSIONS: These data highlight the value of assessing CAR constructs against a panel of cells expressing varying degrees of target tumor antigen as occurs in human tumors. Furthermore, the problem of low antigen density may be overcome by concomitant administration of drugs that inhibit enzymatic shedding of MSLN.
Assuntos
Mesotelioma , Neoplasias Pancreáticas , Receptores de Antígenos Quiméricos , Humanos , Linhagem Celular Tumoral , Imunoterapia Adotiva , Mesotelina , Mesotelioma/terapia , Mesotelioma/patologia , Neoplasias Pancreáticas/terapia , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/metabolismoRESUMO
Malignant pleural mesothelioma (MPM) is an aggressive cancer with limited treatment options and poor prognosis. MPM originates from the mesothelial lining of the pleura. Mesothelin (MSLN) is a glycoprotein expressed at low levels in normal tissues and at high levels in MPM. Many other solid cancers overexpress MSLN, and this is associated with worse survival rates. However, this association has not been found in MPM, and the exact biological role of MSLN in MPM requires further exploration. Here, we discuss the current research on the diagnostic and prognostic value of MSLN in MPM patients. Furthermore, MSLN has become an attractive immunotherapy target in MPM, where better treatment strategies are urgently needed. Several MSLN-targeted monoclonal antibodies, antibody-drug conjugates, immunotoxins, cancer vaccines, and cellular therapies have been tested in the clinical setting. The biological rationale underpinning MSLN-targeted immunotherapies and their potential to improve MPM patient outcomes are reviewed.
RESUMO
Transient receptor potential cation channel subfamily M member 5 (TRPM5) is a nonselective monovalent cation channel activated by intracellular Ca2+ increase. Within the gastrointestinal system, TRPM5 is expressed in the stoma, small intestine, and colon. In the search for a selective agonist of TRPM5 possessing in vivo gastrointestinal prokinetic activity, a high-throughput screening was performed and compound 1 was identified as a promising hit. Hit validation and hit to lead activities led to the discovery of a series of benzo[d]isothiazole derivatives. Among these, compounds 61 and 64 showed nanomolar activity and excellent selectivity (>100-fold) versus related cation channels. The in vivo drug metabolism and pharmacokinetic profile of compound 64 was found to be ideal for a compound acting locally at the intestinal level, with minimal absorption into systemic circulation. Compound 64 was tested in vivo in a mouse motility assay at 100 mg/kg, and demonstrated increased prokinetic activity.
Assuntos
Benzotiazóis/química , Benzotiazóis/farmacologia , Trato Gastrointestinal/metabolismo , Ensaios de Triagem em Larga Escala , Canais de Cátion TRPM/agonistas , Animais , Benzotiazóis/metabolismo , Benzotiazóis/farmacocinética , Desenho de Fármacos , Trato Gastrointestinal/efeitos dos fármacos , Humanos , Masculino , Camundongos , Terapia de Alvo Molecular , Reprodutibilidade dos Testes , Distribuição TecidualRESUMO
Malignant mesothelioma (MM) is a treatment-resistant tumor originating in the mesothelial lining of the pleura or the abdominal cavity with very limited treatment options. More effective therapeutic approaches are urgently needed to improve the poor prognosis of MM patients. Chimeric Antigen Receptor (CAR) T cell therapy has emerged as a novel potential treatment for this incurable solid tumor. The tumor-associated antigen mesothelin (MSLN) is an attractive target for cell therapy in MM, as this antigen is expressed at high levels in the diseased pleura or peritoneum in the majority of MM patients and not (or very modestly) present in healthy tissues. Clinical trials using anti-MSLN CAR T cells in MM have shown that this potential therapeutic is relatively safe. However, efficacy remains modest, likely due to the MM tumor microenvironment (TME), which creates strong immunosuppressive conditions and thus reduces anti-MSLN CAR T cell tumor infiltration, efficacy and persistence. Various approaches to overcome these challenges are reviewed here. They include local (intratumoral) delivery of anti-MSLN CAR T cells, improved CAR design and co-stimulation, and measures to avoid T cell exhaustion. Combination therapies with checkpoint inhibitors as well as oncolytic viruses are also discussed. Preclinical studies have confirmed that increased efficacy of anti-MSLN CAR T cells is within reach and offer hope that this form of cellular immunotherapy may soon improve the prognosis of MM patients.
RESUMO
Polypharmacology approaches may help the discovery of pharmacological tools for the study or the potential treatment of complex and multifactorial diseases as well as for addictions and also smoke cessation. In this frame, following our interest in the development of molecules able to modulate either the endocannabinoid or the dopaminergic system, and given the multiple and reciprocal interconnections between them, we decided to merge the pharmacophoric elements of some of our early leads for identifying new molecules as tools able to modulate both systems. We herein describe the synthesis and biological characterization of compounds 5a-j inspired by the structure of our potent and selective fatty acid amide hydrolase (FAAH) inhibitors (3a-c) and ligands of dopamine D2 or D3 receptor subtypes (4a,b). Notably, the majority of the new molecules showed a nanomolar potency of interaction with the targets of interest. The drug-likeliness of the developed compounds (5a-j) was investigated in silico while hERG affinity, selectivity profile (for some proteins of the endocannabinoid system), cytotoxicity profiles (on fibroblast and astrocytes), and mutagenicity (Ames test) were experimentally determined. Metabolic studies also served to complement the preliminary drug-likeliness profiling for compounds 3a and 5c. Interestingly, after assessing the lack of toxicity for the neuroblastoma cell line (IMR 32), we demonstrated a potential anti-inflammatory profile for 3a and 5c in the same cell line.
Assuntos
Amidoidrolases/antagonistas & inibidores , Dopamina/metabolismo , Endocanabinoides/metabolismo , Amidoidrolases/metabolismo , Ligação Competitiva , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Ligantes , Piperazinas/química , Piperazinas/farmacologia , Pirróis/química , Pirróis/farmacologiaRESUMO
Neutrophils or polymorphonuclear cells (PMN) eliminate bacteria via phagocytosis and/or NETosis. Apart from these conventional roles, PMN also have immune-regulatory functions. They can transdifferentiate and upregulate MHCII as well as ligands for costimulatory receptors which enables them to behave as antigen presenting cells (APC). The initial step for activating T-cells is the formation of an immune synapse between T-cells and antigen-presenting cells. However, the immune synapse that develops at the PMN/T-cell contact zone is as yet hardly investigated due to the non-availability of methods for analysis of large number of PMN interactions. In order to overcome these obstacles, we introduce here a workflow to analyse the immune synapse of primary human PMN and T-cells using multispectral imaging flow cytometry (InFlow microscopy) and super-resolution microscopy. For that purpose, we used CD3 and CD66b as the lineage markers for T-cells and PMN, respectively. Thereafter, we applied and critically discussed various "masks" for identification of T-cell PMN interactions. Using this approach, we found that a small fraction of transdifferentiated PMN (CD66b+CD86high) formed stable PMN/T-cell conjugates. Interestingly, while both CD3 and CD66b accumulation in the immune synapse was dependent on the maturation state of the PMN, only CD3 accumulation was greatly enhanced by the presence of superantigen. The actin cytoskeleton was weakly rearranged at the PMN side on the immune synapse upon contact with a T-cell in the presence of superantigen. A more detailed analysis using super-resolution microscopy (structured-illumination microscopy, SIM) confirmed this finding. Together, we present an InFlow microscopy based approach for the large scale analysis of PMN/T-cell interactions and - combined with SIM - a possibility for an in-depth analysis of protein translocation at the site of interactions.
Assuntos
Células Apresentadoras de Antígenos/metabolismo , Comunicação Celular/imunologia , Citometria de Fluxo/métodos , Citometria por Imagem/métodos , Microscopia/métodos , Linfócitos T/metabolismo , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/imunologia , Citoesqueleto de Actina/ultraestrutura , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/ultraestrutura , Antígenos CD/genética , Antígenos CD/imunologia , Biomarcadores/metabolismo , Complexo CD3/genética , Complexo CD3/imunologia , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/imunologia , Comunicação Celular/genética , Transdiferenciação Celular , Técnicas de Cocultura , Citometria de Fluxo/instrumentação , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/imunologia , Expressão Gênica , Granulócitos/imunologia , Granulócitos/metabolismo , Granulócitos/ultraestrutura , Humanos , Citometria por Imagem/instrumentação , Sinapses Imunológicas/genética , Sinapses Imunológicas/ultraestrutura , Separação Imunomagnética/métodos , Microscopia/instrumentação , Cultura Primária de Células , Linfócitos T/imunologia , Linfócitos T/ultraestruturaRESUMO
A novel series of 1,2,4-triazolyl 5-azaspiro[2.4]heptanes with high affinity and selectivity at the dopamine (DA) D3 receptor (D3R) is described. Some of these compounds also have high selectivity over the hERG channel and were characterized with respect to their pharmacokinetic properties both in vitro and in vivo during lead identification and early lead optimization phases. A few derivatives with overall favorable developability characteristics were selected for further late lead optimization studies.
Assuntos
Heptanos/química , Heptanos/farmacologia , Receptores de Dopamina D3/antagonistas & inibidores , Compostos de Espiro/química , Compostos de Espiro/farmacologia , Animais , Células CHO , Cricetulus , Cristalografia por Raios X , Canais de Potássio Éter-A-Go-Go/metabolismo , Humanos , Modelos Moleculares , Receptores de Dopamina D3/metabolismo , Relação Estrutura-Atividade , Triazóis/química , Triazóis/farmacologiaRESUMO
A novel series of 1,2,4-triazolyl octahydropyrrolo[2,3-b]pyrroles showing high affinity and selectivity at the DA D3 receptor is reported here. Compounds endowed with high selectivity over the hERG channel were identified and their pharmacokinetic properties thoroughly analyzed. A few derivatives with appropriate developability characteristics were selected for further studies and progression along the screening cascade. In particular, derivative 60a, (DA D3 pKi=8.4, DA D2 pKi=6.0 and hERG fpKi=5.2) showed a balanced profile and further refinements are in progress around this molecule.
Assuntos
Pirróis/farmacologia , Receptores de Dopamina D3/antagonistas & inibidores , Triazóis/farmacologia , Animais , Ligação Competitiva/efeitos dos fármacos , Células CHO , Cricetulus , Relação Dose-Resposta a Droga , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Células HEK293 , Humanos , Modelos Moleculares , Estrutura Molecular , Pirróis/síntese química , Pirróis/química , Relação Estrutura-Atividade , Triazóis/síntese química , Triazóis/químicaRESUMO
A series of α7 nicotinic acetylcholine receptor full-agonists with a 1,3,4-oxadiazol-2-amine core has been discovered. Systematic exploration of the structure-activity relationships for both α7 potency and selectivity with respect to interaction with the hERG channel are described. Further profiling led to the identification of compound 22, a potent full agonist showing efficacy in the novel object recognition model of cognition enhancement.
Assuntos
Cognição/efeitos dos fármacos , Agonistas Nicotínicos/farmacologia , Receptores Nicotínicos/efeitos dos fármacos , Animais , Cães , Agonistas Nicotínicos/química , Receptor Nicotínico de Acetilcolina alfa7RESUMO
A series of α7 nicotinic acetylcholine receptor full agonists with a 1,3,4-oxadiazol-2-amine core has been discovered. Early lead 1 was found to have a limited therapeutic index with respect to its potential for cardiovascular side effects. Further optimisation of this series led to the identification of 22 a potent full agonist showing efficacy at a dose of 0.1mg/kg in the novel object recognition model of cognition enhancement. Comparison of 1 with 22 demonstrated the latter to have an improved oral pharmacokinetic profile and cardiovascular therapeutic index.
Assuntos
Cognição/efeitos dos fármacos , Agonistas Nicotínicos/farmacologia , Receptores Nicotínicos/efeitos dos fármacos , Animais , Relação Dose-Resposta a Droga , Coelhos , Receptores Nicotínicos/química , Receptor Nicotínico de Acetilcolina alfa7RESUMO
BACKGROUND AND PURPOSE: Despite growing evidence that inhibition of α6ß2-containing (α6ß2*) nicotinic acetylcholine receptors (nAChRs) may be beneficial for the therapy of tobacco addiction, the lack of good sources of α6ß2*-nAChRs has delayed the discovery of α6ß2-selective antagonists. Our aim was to generate a cell line stably expressing functional nAChRs with α6ß2 properties, to enable pharmacological characterization and the identification of novel α6ß2-selective antagonists. EXPERIMENTAL APPROACH: Different combinations of the α6, ß2, ß3, chimeric α6/3 and mutant ß3(V273S) subunits were transfected in human embryonic kidney cells and tested for activity in a fluorescent imaging plate reader assay. The pharmacology of rat immune-immobilized α6ß2*-nAChRs was determined with ¹²5I-epibatidine binding. KEY RESULTS: Functional channels were detected after co-transfection of α6/3, ß2 and ß3(V273S) subunits, while all other subunit combinations failed to produce agonist-induced responses. Stably expressed α6/3ß2ß3(V273S)-nAChR pharmacology was unique, and clearly distinct from α4ß2-, α3ß4-, α7- and α1ß1δε-nAChRs. Antagonist potencies in inhibiting α6/3ß2ß3(V273S) -nAChRs was similar to their binding affinity for rat native α6ß2*-nAChRs. Agonist affinities for α6ß2*-nAChRs was higher than their potency in activating α6/3ß2ß3(V273S)-nAChRs, but their relative activities were equivalent. Focussed set screening at α6/3ß2ß3(V273S)-nAChRs, followed by cross-screening with the other nAChRs, led to the identification of novel α6ß2-selective antagonists. CONCLUSIONS AND IMPLICATIONS: We generated a mammalian cell line stably expressing nAChRs, with pharmacological properties similar to native α6ß2*-nAChRs, and used it to identify novel non-peptide, low molecular weight, α6ß2-selective antagonists. We also propose a pharmacophore model of α6ß2 antagonists, which offers a starting point for the development of new smoking cessation agents.
Assuntos
Antagonistas Nicotínicos/farmacologia , Receptores Nicotínicos/metabolismo , Animais , Avaliação Pré-Clínica de Medicamentos , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , Masculino , Modelos Moleculares , Mutação , Antagonistas Nicotínicos/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Nicotínicos/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The pharmacophore model of in house potent and selective alpha7 nAChR positive allosteric modulators is reported. The model was used to fish out commercially-available compounds from corporate 3D databases. As a result, novel alpha7 positive modulator chemotypes were identified. A rat full PK profile of a representative compound is also described.
Assuntos
Receptores Nicotínicos/química , Regulação Alostérica , Animais , Linhagem Celular , Bases de Dados Factuais , Humanos , Isoxazóis/química , Isoxazóis/farmacocinética , Microssomos Hepáticos/metabolismo , Modelos Moleculares , Compostos de Fenilureia/química , Compostos de Fenilureia/farmacocinética , Ratos , Receptores Nicotínicos/metabolismo , Relação Estrutura-Atividade , Receptor Nicotínico de Acetilcolina alfa7RESUMO
Chronic administration of antidepressants is required for their efficacy, suggesting the involvement of long-term modifications. As the impact of antidepressant treatment on the brain molecular machinery is not completely understood, we performed a proteomic analysis of rat hippocampus and frontal cortex after chronic treatment with fluoxetine, with an NK1 receptor antagonist, GR205171, and a CRF receptor 1 antagonist, DMP696. After 2D electrophoresis, protein expression levels were compared with both univariate and multivariate statistical analyses and identified by mass spectrometry. All treatments modified levels of actin isoforms, whereas both fluoxetine and GR205171 reduced synapsin II. Fluoxetine treatment increased ERK2 and NP25 and decreased vacuolar ATP synthase. After GR205171 treatment, protein disulphide isomerase A was reduced; dynamin 1 and aldose reductase increased. DMP696 modulated DRP2, pyruvate kinase, LDH and ATP synthase. Although each compound induced a specific pattern of protein modulation, data suggest that antidepressants share the ability of modulating neural plasticity.
Assuntos
Antidepressivos/farmacologia , Fluoxetina/farmacologia , Lobo Frontal/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Antagonistas dos Receptores de Neurocinina-1 , Proteômica , Receptores de Hormônio Liberador da Corticotropina/antagonistas & inibidores , Animais , Western Blotting/métodos , Eletroforese em Gel Bidimensional/métodos , Masculino , Mapeamento de Peptídeos/métodos , Piperidinas/uso terapêutico , Pirazóis/uso terapêutico , Ratos , Ratos Sprague-Dawley , Análise Espectral/métodos , Tetrazóis/uso terapêutico , Triazinas/uso terapêuticoRESUMO
The polymerisation of 3-aminophenylboronic acid (APBA) in aqueous environment has been used for the open tubular modification of capillary electrophoresis (CE) capillaries. Being poly-APBA endowed with boronic acid, aromatic rings and secondary amines groups, it posses a variety of functional groups affecting selectivity. Diastereoisomers (e.g. ascorbic and isoascorbic acid) and proteins (e.g. haemoglobins) were successfully separated onto poly-APBA column, by means of a combination of electrophoresis and open tubular electrochromatography. The mechanism of selection was investigated: results indicate an interplay between enhancing or silencing the contribution of the protonable functionalities (amino groups, boronic acid). The properties of APBA polymer coating make it attractive for CE separation and for further application in affinity separations and chip technologies.
Assuntos
Ácidos Borônicos/química , Eletroforese Capilar/métodos , Glicoproteínas/isolamento & purificação , EstereoisomerismoRESUMO
Extending the previous preliminary study on the construction of a capillary electrophoresis (CE)/sensor for the detection of reducing analytes, we focus the interest on the simultaneous detection of redox active species, which are important indicators of the oxidative damage in tissues, of food preservation, and of pollution. The CE/sensor was built by modifying the detector-portion of the capillary with the redox-sensitive polymer polyaniline (PANI). The analyte is detected by monitoring the changes in optical absorption of the PANI film. The CE/sensor was tested, with good results, with ascorbic acid, glutathione (GSH), as well as with compounds with very close similarity (ascorbic and isoascorbic acid). The kinetics of oxidation and reduction of PANI were evaluated. Further a PANI/CE-biological sensor was developed by coupling an enzyme, glucose oxidase (GOD), to the PANI-modified portion of the capillary. The stability of the immobilized GOD and the sensitivity of the CE/biosensor were studied, by using glucose as test analyte in concentrations within the physiological range. The results indicate that the CE/biosensor had good stability (more than 75% of original activity retained after 30 operational days), manufacturing reproducibility and a sensing range convenient for monitoring physiological glucose (1-24 mM).
Assuntos
Técnicas Biossensoriais/instrumentação , Eletroforese Capilar/instrumentação , Glucose Oxidase , Glucose/análise , Compostos de Anilina , Ácido Ascórbico/análise , Técnicas Biossensoriais/normas , Enzimas Imobilizadas/metabolismo , Glucose Oxidase/metabolismo , Glutationa/análise , Cinética , OxirreduçãoRESUMO
A CE/biosensor for measuring ascorbic acid was developed by coupling a polyaniline optical sensor and capillary electrophoresis (CE). The capillary column was partially modified with a thin film of polyaniline redox sensitive material. Ascorbic acid was detected by monitoring the changes in optical absorbance occurring to the polyaniline film upon the reduction reaction. The sensor response (change in optical absorbance at 650 nm) is proportional to the concentration of ascorbic acid over a range of 2.5-250 mg/L and the response range has shown a clear dependence on the characteristics of the polymerized film. High specificity and sensitivity of the present method, low sample consumption, short times of response (ca. 2 min) and the reproducibility of the results demonstrate that the CE/polyaniline-sensor could be further employed in the study of the relation between the content of L-ascorbic acid in body fluids and clinical parameters, e.g., cell ageing.