5′-Substituted Amiloride Derivatives as Allosteric Modulators Binding in the Sodium Ion Pocket of the Adenosine A_2A Receptor

The sodium ion site is an allosteric site conserved among many G protein-coupled receptors (GPCRs). Amiloride <b>1</b> and 5-(<i>N</i>,<i>N</i>-hexamethylene)­amiloride <b>2</b> (HMA) supposedly bind in this sodium ion site and can influence orthosteric ligand binding. The availability of a high-resolution X-ray crystal structure of the human adenosine A_2A receptor (hA_2AAR), in which the allosteric sodium ion site was elucidated, makes it an appropriate model receptor for investigating the allosteric site. In this study, we report the synthesis and evaluation of novel 5′-substituted amiloride derivatives as hA_2AAR allosteric antagonists. The potency of the amiloride derivatives was assessed by their ability to displace orthosteric radioligand [³H]­4-(2-((7-amino-2-(furan-2-yl)-[1,2,4]­triazolo­[1,5-<i>a</i>]-[1,3,5]­triazin-5-yl)­amino)­ethyl)­phenol ([³H]­ZM-241,385) from both the wild-type and sodium ion site W246A mutant hA_2AAR. 4-Ethoxy­phenethyl-substituted amiloride <b>12l</b> was found to be more potent than both amiloride and HMA, and the shift in potency between the wild-type and mutated receptor confirmed its likely binding to the sodium ion site

5′-Substituted Amiloride Derivatives as Allosteric Modulators Binding in the Sodium Ion Pocket of the Adenosine A_2A Receptor

The sodium ion site is an allosteric site conserved among many G protein-coupled receptors (GPCRs). Amiloride <b>1</b> and 5-(<i>N</i>,<i>N</i>-hexamethylene)­amiloride <b>2</b> (HMA) supposedly bind in this sodium ion site and can influence orthosteric ligand binding. The availability of a high-resolution X-ray crystal structure of the human adenosine A_2A receptor (hA_2AAR), in which the allosteric sodium ion site was elucidated, makes it an appropriate model receptor for investigating the allosteric site. In this study, we report the synthesis and evaluation of novel 5′-substituted amiloride derivatives as hA_2AAR allosteric antagonists. The potency of the amiloride derivatives was assessed by their ability to displace orthosteric radioligand [³H]­4-(2-((7-amino-2-(furan-2-yl)-[1,2,4]­triazolo­[1,5-<i>a</i>]-[1,3,5]­triazin-5-yl)­amino)­ethyl)­phenol ([³H]­ZM-241,385) from both the wild-type and sodium ion site W246A mutant hA_2AAR. 4-Ethoxy­phenethyl-substituted amiloride <b>12l</b> was found to be more potent than both amiloride and HMA, and the shift in potency between the wild-type and mutated receptor confirmed its likely binding to the sodium ion site

Image_3_Metastatic phenotype and immunosuppressive tumour microenvironment in pancreatic ductal adenocarcinoma: Key role of the urokinase plasminogen activator (PLAU).jpeg

BackgroundPrevious studies have revealed the role of dysregulated urokinase plasminogen activator (encoded by PLAU) expression and activity in several pathways associated with cancer progression. However, systematic investigation into the association of PLAU expression with factors that modulate PDAC (pancreatic ductal adenocarcinoma) progression is lacking, such as those affecting stromal (pancreatic stellate cell, PSC)-cancer cell interactions, tumour immunity, PDAC subtypes and clinical outcomes from potential PLAU inhibition.MethodsThis study used an integrated bioinformatics approach to identify prognostic markers correlated with PLAU expression using different transcriptomics, proteomics, and clinical data sets. We then determined the association of dysregulated PLAU and correlated signatures with oncogenic pathways, metastatic phenotypes, stroma, immunosuppressive tumour microenvironment (TME) and clinical outcome. Finally, using an in vivo orthotopic model of pancreatic cancer, we confirmed the predicted effect of inhibiting PLAU on tumour growth and metastasis.ResultsOur analyses revealed that PLAU upregulation is not only associated with numerous other prognostic markers but also associated with the activation of various oncogenic signalling pathways, aggressive phenotypes relevant to PDAC growth and metastasis, such as proliferation, epithelial-mesenchymal transition (EMT), stemness, hypoxia, extracellular cell matrix (ECM) degradation, upregulation of stromal signatures, and immune suppression in the tumour microenvironment (TME). Moreover, the upregulation of PLAU was directly connected with signalling pathways known to mediate PSC-cancer cell interactions. Furthermore, PLAU upregulation was associated with the aggressive basal/squamous phenotype of PDAC and significantly reduced overall survival, indicating that this subset of patients may benefit from therapeutic interventions to inhibit PLAU activity. Our studies with a clinically relevant orthotopic pancreatic model showed that even short-term PLAU inhibition is sufficient to significantly halt tumour growth and, importantly, eliminate visible metastasis.ConclusionElevated PLAU correlates with increased aggressive phenotypes, stromal score, and immune suppression in PDAC. PLAU upregulation is also closely associated with the basal subtype type of PDAC; patients with this subtype are at high risk of mortality from the disease and may benefit from therapeutic targeting of PLAU.</p

Image_8_Metastatic phenotype and immunosuppressive tumour microenvironment in pancreatic ductal adenocarcinoma: Key role of the urokinase plasminogen activator (PLAU).tif

BackgroundPrevious studies have revealed the role of dysregulated urokinase plasminogen activator (encoded by PLAU) expression and activity in several pathways associated with cancer progression. However, systematic investigation into the association of PLAU expression with factors that modulate PDAC (pancreatic ductal adenocarcinoma) progression is lacking, such as those affecting stromal (pancreatic stellate cell, PSC)-cancer cell interactions, tumour immunity, PDAC subtypes and clinical outcomes from potential PLAU inhibition.MethodsThis study used an integrated bioinformatics approach to identify prognostic markers correlated with PLAU expression using different transcriptomics, proteomics, and clinical data sets. We then determined the association of dysregulated PLAU and correlated signatures with oncogenic pathways, metastatic phenotypes, stroma, immunosuppressive tumour microenvironment (TME) and clinical outcome. Finally, using an in vivo orthotopic model of pancreatic cancer, we confirmed the predicted effect of inhibiting PLAU on tumour growth and metastasis.ResultsOur analyses revealed that PLAU upregulation is not only associated with numerous other prognostic markers but also associated with the activation of various oncogenic signalling pathways, aggressive phenotypes relevant to PDAC growth and metastasis, such as proliferation, epithelial-mesenchymal transition (EMT), stemness, hypoxia, extracellular cell matrix (ECM) degradation, upregulation of stromal signatures, and immune suppression in the tumour microenvironment (TME). Moreover, the upregulation of PLAU was directly connected with signalling pathways known to mediate PSC-cancer cell interactions. Furthermore, PLAU upregulation was associated with the aggressive basal/squamous phenotype of PDAC and significantly reduced overall survival, indicating that this subset of patients may benefit from therapeutic interventions to inhibit PLAU activity. Our studies with a clinically relevant orthotopic pancreatic model showed that even short-term PLAU inhibition is sufficient to significantly halt tumour growth and, importantly, eliminate visible metastasis.ConclusionElevated PLAU correlates with increased aggressive phenotypes, stromal score, and immune suppression in PDAC. PLAU upregulation is also closely associated with the basal subtype type of PDAC; patients with this subtype are at high risk of mortality from the disease and may benefit from therapeutic targeting of PLAU.</p

Image_1_Metastatic phenotype and immunosuppressive tumour microenvironment in pancreatic ductal adenocarcinoma: Key role of the urokinase plasminogen activator (PLAU).jpeg

BackgroundPrevious studies have revealed the role of dysregulated urokinase plasminogen activator (encoded by PLAU) expression and activity in several pathways associated with cancer progression. However, systematic investigation into the association of PLAU expression with factors that modulate PDAC (pancreatic ductal adenocarcinoma) progression is lacking, such as those affecting stromal (pancreatic stellate cell, PSC)-cancer cell interactions, tumour immunity, PDAC subtypes and clinical outcomes from potential PLAU inhibition.MethodsThis study used an integrated bioinformatics approach to identify prognostic markers correlated with PLAU expression using different transcriptomics, proteomics, and clinical data sets. We then determined the association of dysregulated PLAU and correlated signatures with oncogenic pathways, metastatic phenotypes, stroma, immunosuppressive tumour microenvironment (TME) and clinical outcome. Finally, using an in vivo orthotopic model of pancreatic cancer, we confirmed the predicted effect of inhibiting PLAU on tumour growth and metastasis.ResultsOur analyses revealed that PLAU upregulation is not only associated with numerous other prognostic markers but also associated with the activation of various oncogenic signalling pathways, aggressive phenotypes relevant to PDAC growth and metastasis, such as proliferation, epithelial-mesenchymal transition (EMT), stemness, hypoxia, extracellular cell matrix (ECM) degradation, upregulation of stromal signatures, and immune suppression in the tumour microenvironment (TME). Moreover, the upregulation of PLAU was directly connected with signalling pathways known to mediate PSC-cancer cell interactions. Furthermore, PLAU upregulation was associated with the aggressive basal/squamous phenotype of PDAC and significantly reduced overall survival, indicating that this subset of patients may benefit from therapeutic interventions to inhibit PLAU activity. Our studies with a clinically relevant orthotopic pancreatic model showed that even short-term PLAU inhibition is sufficient to significantly halt tumour growth and, importantly, eliminate visible metastasis.ConclusionElevated PLAU correlates with increased aggressive phenotypes, stromal score, and immune suppression in PDAC. PLAU upregulation is also closely associated with the basal subtype type of PDAC; patients with this subtype are at high risk of mortality from the disease and may benefit from therapeutic targeting of PLAU.</p

Image_5_Metastatic phenotype and immunosuppressive tumour microenvironment in pancreatic ductal adenocarcinoma: Key role of the urokinase plasminogen activator (PLAU).tif

BackgroundPrevious studies have revealed the role of dysregulated urokinase plasminogen activator (encoded by PLAU) expression and activity in several pathways associated with cancer progression. However, systematic investigation into the association of PLAU expression with factors that modulate PDAC (pancreatic ductal adenocarcinoma) progression is lacking, such as those affecting stromal (pancreatic stellate cell, PSC)-cancer cell interactions, tumour immunity, PDAC subtypes and clinical outcomes from potential PLAU inhibition.MethodsThis study used an integrated bioinformatics approach to identify prognostic markers correlated with PLAU expression using different transcriptomics, proteomics, and clinical data sets. We then determined the association of dysregulated PLAU and correlated signatures with oncogenic pathways, metastatic phenotypes, stroma, immunosuppressive tumour microenvironment (TME) and clinical outcome. Finally, using an in vivo orthotopic model of pancreatic cancer, we confirmed the predicted effect of inhibiting PLAU on tumour growth and metastasis.ResultsOur analyses revealed that PLAU upregulation is not only associated with numerous other prognostic markers but also associated with the activation of various oncogenic signalling pathways, aggressive phenotypes relevant to PDAC growth and metastasis, such as proliferation, epithelial-mesenchymal transition (EMT), stemness, hypoxia, extracellular cell matrix (ECM) degradation, upregulation of stromal signatures, and immune suppression in the tumour microenvironment (TME). Moreover, the upregulation of PLAU was directly connected with signalling pathways known to mediate PSC-cancer cell interactions. Furthermore, PLAU upregulation was associated with the aggressive basal/squamous phenotype of PDAC and significantly reduced overall survival, indicating that this subset of patients may benefit from therapeutic interventions to inhibit PLAU activity. Our studies with a clinically relevant orthotopic pancreatic model showed that even short-term PLAU inhibition is sufficient to significantly halt tumour growth and, importantly, eliminate visible metastasis.ConclusionElevated PLAU correlates with increased aggressive phenotypes, stromal score, and immune suppression in PDAC. PLAU upregulation is also closely associated with the basal subtype type of PDAC; patients with this subtype are at high risk of mortality from the disease and may benefit from therapeutic targeting of PLAU.</p

Image_2_Metastatic phenotype and immunosuppressive tumour microenvironment in pancreatic ductal adenocarcinoma: Key role of the urokinase plasminogen activator (PLAU).tif

BackgroundPrevious studies have revealed the role of dysregulated urokinase plasminogen activator (encoded by PLAU) expression and activity in several pathways associated with cancer progression. However, systematic investigation into the association of PLAU expression with factors that modulate PDAC (pancreatic ductal adenocarcinoma) progression is lacking, such as those affecting stromal (pancreatic stellate cell, PSC)-cancer cell interactions, tumour immunity, PDAC subtypes and clinical outcomes from potential PLAU inhibition.MethodsThis study used an integrated bioinformatics approach to identify prognostic markers correlated with PLAU expression using different transcriptomics, proteomics, and clinical data sets. We then determined the association of dysregulated PLAU and correlated signatures with oncogenic pathways, metastatic phenotypes, stroma, immunosuppressive tumour microenvironment (TME) and clinical outcome. Finally, using an in vivo orthotopic model of pancreatic cancer, we confirmed the predicted effect of inhibiting PLAU on tumour growth and metastasis.ResultsOur analyses revealed that PLAU upregulation is not only associated with numerous other prognostic markers but also associated with the activation of various oncogenic signalling pathways, aggressive phenotypes relevant to PDAC growth and metastasis, such as proliferation, epithelial-mesenchymal transition (EMT), stemness, hypoxia, extracellular cell matrix (ECM) degradation, upregulation of stromal signatures, and immune suppression in the tumour microenvironment (TME). Moreover, the upregulation of PLAU was directly connected with signalling pathways known to mediate PSC-cancer cell interactions. Furthermore, PLAU upregulation was associated with the aggressive basal/squamous phenotype of PDAC and significantly reduced overall survival, indicating that this subset of patients may benefit from therapeutic interventions to inhibit PLAU activity. Our studies with a clinically relevant orthotopic pancreatic model showed that even short-term PLAU inhibition is sufficient to significantly halt tumour growth and, importantly, eliminate visible metastasis.ConclusionElevated PLAU correlates with increased aggressive phenotypes, stromal score, and immune suppression in PDAC. PLAU upregulation is also closely associated with the basal subtype type of PDAC; patients with this subtype are at high risk of mortality from the disease and may benefit from therapeutic targeting of PLAU.</p

Image_7_Metastatic phenotype and immunosuppressive tumour microenvironment in pancreatic ductal adenocarcinoma: Key role of the urokinase plasminogen activator (PLAU).tif

BackgroundPrevious studies have revealed the role of dysregulated urokinase plasminogen activator (encoded by PLAU) expression and activity in several pathways associated with cancer progression. However, systematic investigation into the association of PLAU expression with factors that modulate PDAC (pancreatic ductal adenocarcinoma) progression is lacking, such as those affecting stromal (pancreatic stellate cell, PSC)-cancer cell interactions, tumour immunity, PDAC subtypes and clinical outcomes from potential PLAU inhibition.MethodsThis study used an integrated bioinformatics approach to identify prognostic markers correlated with PLAU expression using different transcriptomics, proteomics, and clinical data sets. We then determined the association of dysregulated PLAU and correlated signatures with oncogenic pathways, metastatic phenotypes, stroma, immunosuppressive tumour microenvironment (TME) and clinical outcome. Finally, using an in vivo orthotopic model of pancreatic cancer, we confirmed the predicted effect of inhibiting PLAU on tumour growth and metastasis.ResultsOur analyses revealed that PLAU upregulation is not only associated with numerous other prognostic markers but also associated with the activation of various oncogenic signalling pathways, aggressive phenotypes relevant to PDAC growth and metastasis, such as proliferation, epithelial-mesenchymal transition (EMT), stemness, hypoxia, extracellular cell matrix (ECM) degradation, upregulation of stromal signatures, and immune suppression in the tumour microenvironment (TME). Moreover, the upregulation of PLAU was directly connected with signalling pathways known to mediate PSC-cancer cell interactions. Furthermore, PLAU upregulation was associated with the aggressive basal/squamous phenotype of PDAC and significantly reduced overall survival, indicating that this subset of patients may benefit from therapeutic interventions to inhibit PLAU activity. Our studies with a clinically relevant orthotopic pancreatic model showed that even short-term PLAU inhibition is sufficient to significantly halt tumour growth and, importantly, eliminate visible metastasis.ConclusionElevated PLAU correlates with increased aggressive phenotypes, stromal score, and immune suppression in PDAC. PLAU upregulation is also closely associated with the basal subtype type of PDAC; patients with this subtype are at high risk of mortality from the disease and may benefit from therapeutic targeting of PLAU.</p

DataSheet_1_Metastatic phenotype and immunosuppressive tumour microenvironment in pancreatic ductal adenocarcinoma: Key role of the urokinase plasminogen activator (PLAU).xlsx

BackgroundPrevious studies have revealed the role of dysregulated urokinase plasminogen activator (encoded by PLAU) expression and activity in several pathways associated with cancer progression. However, systematic investigation into the association of PLAU expression with factors that modulate PDAC (pancreatic ductal adenocarcinoma) progression is lacking, such as those affecting stromal (pancreatic stellate cell, PSC)-cancer cell interactions, tumour immunity, PDAC subtypes and clinical outcomes from potential PLAU inhibition.MethodsThis study used an integrated bioinformatics approach to identify prognostic markers correlated with PLAU expression using different transcriptomics, proteomics, and clinical data sets. We then determined the association of dysregulated PLAU and correlated signatures with oncogenic pathways, metastatic phenotypes, stroma, immunosuppressive tumour microenvironment (TME) and clinical outcome. Finally, using an in vivo orthotopic model of pancreatic cancer, we confirmed the predicted effect of inhibiting PLAU on tumour growth and metastasis.ResultsOur analyses revealed that PLAU upregulation is not only associated with numerous other prognostic markers but also associated with the activation of various oncogenic signalling pathways, aggressive phenotypes relevant to PDAC growth and metastasis, such as proliferation, epithelial-mesenchymal transition (EMT), stemness, hypoxia, extracellular cell matrix (ECM) degradation, upregulation of stromal signatures, and immune suppression in the tumour microenvironment (TME). Moreover, the upregulation of PLAU was directly connected with signalling pathways known to mediate PSC-cancer cell interactions. Furthermore, PLAU upregulation was associated with the aggressive basal/squamous phenotype of PDAC and significantly reduced overall survival, indicating that this subset of patients may benefit from therapeutic interventions to inhibit PLAU activity. Our studies with a clinically relevant orthotopic pancreatic model showed that even short-term PLAU inhibition is sufficient to significantly halt tumour growth and, importantly, eliminate visible metastasis.ConclusionElevated PLAU correlates with increased aggressive phenotypes, stromal score, and immune suppression in PDAC. PLAU upregulation is also closely associated with the basal subtype type of PDAC; patients with this subtype are at high risk of mortality from the disease and may benefit from therapeutic targeting of PLAU.</p

Image_4_Metastatic phenotype and immunosuppressive tumour microenvironment in pancreatic ductal adenocarcinoma: Key role of the urokinase plasminogen activator (PLAU).tif

BackgroundPrevious studies have revealed the role of dysregulated urokinase plasminogen activator (encoded by PLAU) expression and activity in several pathways associated with cancer progression. However, systematic investigation into the association of PLAU expression with factors that modulate PDAC (pancreatic ductal adenocarcinoma) progression is lacking, such as those affecting stromal (pancreatic stellate cell, PSC)-cancer cell interactions, tumour immunity, PDAC subtypes and clinical outcomes from potential PLAU inhibition.MethodsThis study used an integrated bioinformatics approach to identify prognostic markers correlated with PLAU expression using different transcriptomics, proteomics, and clinical data sets. We then determined the association of dysregulated PLAU and correlated signatures with oncogenic pathways, metastatic phenotypes, stroma, immunosuppressive tumour microenvironment (TME) and clinical outcome. Finally, using an in vivo orthotopic model of pancreatic cancer, we confirmed the predicted effect of inhibiting PLAU on tumour growth and metastasis.ResultsOur analyses revealed that PLAU upregulation is not only associated with numerous other prognostic markers but also associated with the activation of various oncogenic signalling pathways, aggressive phenotypes relevant to PDAC growth and metastasis, such as proliferation, epithelial-mesenchymal transition (EMT), stemness, hypoxia, extracellular cell matrix (ECM) degradation, upregulation of stromal signatures, and immune suppression in the tumour microenvironment (TME). Moreover, the upregulation of PLAU was directly connected with signalling pathways known to mediate PSC-cancer cell interactions. Furthermore, PLAU upregulation was associated with the aggressive basal/squamous phenotype of PDAC and significantly reduced overall survival, indicating that this subset of patients may benefit from therapeutic interventions to inhibit PLAU activity. Our studies with a clinically relevant orthotopic pancreatic model showed that even short-term PLAU inhibition is sufficient to significantly halt tumour growth and, importantly, eliminate visible metastasis.ConclusionElevated PLAU correlates with increased aggressive phenotypes, stromal score, and immune suppression in PDAC. PLAU upregulation is also closely associated with the basal subtype type of PDAC; patients with this subtype are at high risk of mortality from the disease and may benefit from therapeutic targeting of PLAU.</p