bioRxiv (Cold Spring Harbor Laboratory), May 10, 2023
The sperm-specific channel CatSper (cation channel of sperm) controls the intracellular Ca 2+ con... more The sperm-specific channel CatSper (cation channel of sperm) controls the intracellular Ca 2+ concentration ([Ca 2+ ]i) and plays an essential role in sperm function. It is mainly activated by the steroid progesterone (P4) but is also promiscuously activated by a wide range of synthetic and physiological compounds. These compounds include diverse steroids whose action on the channel is so far still controversial. To investigate the effect of these compounds on CatSper and sperm function, we developed a high-throughput-screening (HTS) assay to measure changes in [Ca 2+ ]i in human sperm and screened 1,280 approved and off-patent drugs including 90 steroids from the Prestwick chemical library. More than half of the steroids tested (53%) induced an increase in [Ca 2+ ]i and reduced the P4-induced Ca 2+ influx in human sperm in a dose-dependent manner. Ten of the most potent steroids (activating and inhibiting) were selected for a detailed analysis of their action on CatSper and their ability to act on sperm motility, acrosomal exocytosis (AR), and penetration in viscous media. We found that these steroids show an inhibitory effect on P4 but not on prostaglandin E1-induced CatSper activation, suggesting that they compete for the same binding site as P4. Pregnenolone, dydrogesterone, epiandrosterone, nandrolone, and dehydroepiandrosterone acetate (DHEA) were found to activate CatSper at physiological concentrations. Stanozolol, epiandrosterone, and pregnenolone induced AR similarly to P4, whereas stanozolol and estropipate induced an increase in sperm penetration into viscous medium. Furthermore, using a hybrid approach integrating pharmacophore analysis and statistical modelling, we were able to screen in silico for steroids that can activate the channel and define the physicochemical and structural properties required for a steroid to exhibit agonist activity against CatSper. Overall, our results indicate that not only physiological but also synthetic steroids can modulate the activity of CatSper with varying potency and affect human sperm functions in vitro. .
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
The nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) multi-subunit complex is a highl... more The nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) multi-subunit complex is a highly abundant and central source of reactive oxygen species. NOX2 is a key enzyme of the innate immune system involved in antibacterial response, but excessive NOX2 activity is involved in oxidative stress and inflammation in many diseases. Inhibition of NOX2 has great potential as a therapeutic strategy. An intriguing pharmacological approach for inhibiting NOX2 is to target the p47phox subunit and thereby block the protein-protein interaction with p22phox, whereby assembling and activation of NOX2 is prevented. However, the shallow binding pocket of p47phox makes it difficult to develop drug-like p47phox/p22phox inhibitors. Recently, the small molecule LMH001 was reported to inhibit the p47phox/p22phox interaction, reduce endothelial NOX2 activity, and protect mice from angiotensin II-induced vascular oxidative stress. These noteworthy results could have significant impact on the field of NOX2 pharmacology, as specific and efficient inhibitors are scarce. Here, we synthesized and tested LMH001 to have it available as a positive control. We established a robust synthetic route for providing LMH001, but subsequently we experienced that LMH001 is chemically unstable in aqueous buffer. In addition, neither LMH001 nor its breakdown products were able to inhibit the p47phox/ p22phox interaction in a non-cellular fluorescence polarization assay. However, LHM001 was a weak inhibitor of NOX2 in a functional cell assay, but with same low potency as one of its breakdown products. These findings question the activity and suggested mechanism of LMH001 and constitute important information for other researchers interested in chemical probes for studying NOX2 biology.
The sperm-specific channel CatSper (cation channel ofsperm) controls the intracellular Ca2+concen... more The sperm-specific channel CatSper (cation channel ofsperm) controls the intracellular Ca2+concentration ([Ca2+]i) and plays an essential role in sperm function. It is mainly activated by the steroid progesterone (P4) but is also promiscuously activated by a wide range of synthetic and physiological compounds. These compounds include diverse steroids whose action on the channel is so far still controversial. To investigate the effect of these compounds on CatSper and sperm function, we developed a high-throughput-screening (HTS) assay to measure changes in [Ca2+]iin human sperm and screened 1,280 approved and off-patent drugs including 90 steroids from the Prestwick chemical library. More than half of the steroids tested (53%) induced an increase in [Ca2+]iand reduced the P4-induced Ca2+influx in human sperm in a dose-dependent manner. Ten of the most potent steroids (activating and inhibiting) were selected for a detailed analysis of their action on CatSper and their ability to act...
The nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) multi-subunit complex is a highl... more The nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) multi-subunit complex is a highly abundant and central source of reactive oxygen species. NOX2 is a key enzyme of the innate immune system involved in antibacterial response, but excessive NOX2 activity is involved in oxidative stress and inflammation in many diseases. Inhibition of NOX2 has great potential as a therapeutic strategy. An intriguing pharmacological approach for inhibiting NOX2 is to target the p47phox subunit and thereby block the protein-protein interaction with p22phox, whereby assembling and activation of NOX2 is prevented. However, the shallow binding pocket of p47phox makes it difficult to develop drug-like p47phox/p22phox inhibitors. Recently, the small molecule LMH001 was reported to inhibit the p47phox/p22phox interaction, reduce endothelial NOX2 activity, and protect mice from angiotensin II-induced vascular oxidative stress. These noteworthy results could have significant impact on the field of...
Hydrogen peroxide (H2O2) is a key defense component of host-microbe interaction. However, H2O2 co... more Hydrogen peroxide (H2O2) is a key defense component of host-microbe interaction. However, H2O2 concentrations generated by immune cells or epithelia are usually insufficient for bacterial killing and rather modulate bacterial responses. Here, we investigated the impact of sublethal H2O2 concentration on gene expression of E. coli BW25113 after 10 and 60 min of exposure. RNA-seq analysis revealed that approximately 12% of bacterial genes were strongly dysregulated 10 min following exposure to 2.5 mM H2O2. H2O2 exposure led to the activation of a specific antioxidant response and a general stress response. The latter was characterized by a transient down-regulation of genes involved in general metabolism, such as nucleic acid biosynthesis and translation, with a striking and coordinated down-regulation of genes involved in ribosome formation, and a sustained up-regulation of the SOS response. We confirmed the rapid transient and specific response mediated by the transcription factor O...
Background: The SARS-CoV-2 pandemic was particularly devastating for elderly people, and the unde... more Background: The SARS-CoV-2 pandemic was particularly devastating for elderly people, and the underlying mechanisms of the disease are still poorly understood. In this study, we investigated fusion inhibitory antibodies (fiAbs) in elderly and younger COVID-19 patients and analyzed predictive factors for their occurrence. Methods: Data and samples were collected in two cohorts of hospitalized patients. A fusion assay of SARS-CoV-2 spike-expressing cells with ACE2-expressing cells was used to quantify fiAbs in the serum of patients. Results: A total of 108 patients (52 elderly (mean age 85 ± 7 years); 56 young (mean age 52 ± 10 years)) were studied. The concentrations of fiAbs were lower in geriatric patients, as evidenced at high serum dilutions (1/512). The association between fiAbs and anti-Spike Ig levels was weak (correlation coefficient < 0.3), but statistically significant. Variables associated with fusion were the delay between the onset of symptoms and testing (HR = −2.69; ...
Several lines of evidence support the hypothesis that c-Jun N-terminal kinases (JNKs) play a crit... more Several lines of evidence support the hypothesis that c-Jun N-terminal kinases (JNKs) play a critical role in a wide range of disease states including cell death (apoptosis)-related and inflammatory disorders (epilepsy, brain, heart and renal ischemia, neurodegenerative diseases, multiple sclerosis, rheumatoid arthritis, and inflammatory bowel syndrome). The screening of a compound collection led to the identification of a 2-(benzoylaminomethyl)thiophene sulfonamide (AS004509, compound I) as a potent and selective JNK inhibitor. Chemistry and structure--activity relationship (SAR) studies performed around this novel kinase-inhibiting motif indicated that the left and central parts of the molecule were instrumental to maintaining potency at the enzyme. Accordingly, we investigated the JNK-inhibiting properties of a number of variants of the right-hand moiety of the molecule, which led to the identification of 2-(benzoylaminomethyl)thiophene sulfonamide benzotriazole (AS600292, compou...
Age-related hearing (ARHL) loss affects a large part of the human population with a major impact ... more Age-related hearing (ARHL) loss affects a large part of the human population with a major impact on our aging societies. Yet, underlying mechanisms are not understood, and no validated therapy or prevention exists. NADPH oxidases (NOX), are important sources of reactive oxygen species (ROS) in the cochlea and might therefore be involved in the pathogenesis of ARHL. Here we investigate ARHL in a mouse model. Wild type mice showed early loss of hearing and cochlear integrity, while animals deficient in the NOX subunit p22 phox remained unaffected up to six months. Genes of the excitatory pathway were down-regulated in p22 phox-deficient auditory neurons. Our results demonstrate that NOX activity leads to upregulation of genes of the excitatory pathway, to excitotoxic cochlear damage, and ultimately to ARHL. In the absence of functional NOXs, aging mice conserve hearing and cochlear morphology. Our study offers new insights into pathomechanisms and future therapeutic targets of ARHL.
Several lines of evidence support the hypothesis that c-Jun N-terminal kinases (JNKs) play a crit... more Several lines of evidence support the hypothesis that c-Jun N-terminal kinases (JNKs) play a critical role in a wide range of disease states including cell death (apoptosis)-related and inflammatory disorders (epilepsy, brain, heart and renal ischemia, neurodegenerative diseases, multiple sclerosis, rheumatoid arthritis, and inflammatory bowel syndrome). The screening of a compound collection led to the identification of a 2-(benzoylaminomethyl)thiophene sulfonamide (AS004509, compound I) as a potent and selective JNK inhibitor. Chemistry and structure--activity relationship (SAR) studies performed around this novel kinase-inhibiting motif indicated that the left and central parts of the molecule were instrumental to maintaining potency at the enzyme. Accordingly, we investigated the JNK-inhibiting properties of a number of variants of the right-hand moiety of the molecule, which led to the identification of 2-(benzoylaminomethyl)thiophene sulfonamide benzotriazole (AS600292, compou...
In the field of screening in general and especially in the kinase area, taking into consideration... more In the field of screening in general and especially in the kinase area, taking into consideration throughput and cost, fluorescence-and luminescence-based assays have been developed as alternatives to radioactivity-based assays. However, fluorescence-based technologies are not devoid of pitfalls. One of the main problems is interference from autofluorescent compounds and the incidence of false-positives as exemplified here with a fluorescence polarization (FP)-based assay. Using the scintillation proximity assay as the in-house standard, we assessed several alternatives to radioactive methods, namely, the amplified luminescent proximity homeogeneous assay screen (ALPHAScreen™, Perkin-Elmer Life Sciences, Boston, MA), enzyme fragment complementation, FP, and nanofluidics-based fluorescence intensity. Data comparing the sensitivity, robustness, relative sensitivity to autofluorescent compounds, enzyme consumption, and relative costs of each assay for one common kinase are presented. Results obtained seem to favor the nanofluidics mobility shift assay as a method of choice, followed by the direct FP approach, using generic high-molecularweight phosphate group-binders.
Phenazopyridine is a widely used drug against urinary tract pain. The compound has also been show... more Phenazopyridine is a widely used drug against urinary tract pain. The compound has also been shown to enhance neural differentiation of pluripotent stem cells. However, its mechanism of action is not understood. Based on its chemical structure, we hypothesized that phenazopyridine could be a kinase inhibitor. Phenazopyridine was investigated in the following experimental systems: 1) activity of kinases in pluripotent stem cells; 2) binding to recombinant kinases, and 3) functional impact on pluripotent stem cells. Upon addition to pluripotent stem cells, phenazopyridine induced changes in kinase activities, particularly involving Mitogen-Activated Protein Kinases, Cyclin-Dependent Kinases, and AKT pathway kinases. To identify the primary targets of phenazopyridine, we screened its interactions with 401 human kinases. Dose-inhibition curves showed that three of these kinases interacted with phenazopyridine with sub-micromolar binding affinities: cyclin-G-associated kinase, and the tw...
Cytokines induce the expression of E-selectin, VCAM-1, and ICAM-1 on human umbilical vein endothe... more Cytokines induce the expression of E-selectin, VCAM-1, and ICAM-1 on human umbilical vein endothelial cells (HUVECs). We show that expression of these surface proteins is differently affected by cAMP. Increased cAMP levels decrease E-selectin and VCAM-1 but increase ICAM-1 expression. We demonstrate by mRNA half-life analysis and nuclear run-on assays that the cAMP repression of E-selectin occurs at the transcription level. This effect is abolished by protein kinase A inhibition, suggesting that repression is mediated by protein kinase A-driven phosphorylation. We found that a minimal E-selectin promoter sequence necessary to confer cytokine inducibility is also sufficient to mimic the cAMP effect in transfected HUVECs. Previously we characterized two regions (NF-kappa B and NF-ELAM1) of the minimal promoter that bind transcription factors necessary for E-selectin induction, Increased cAMP did not alter the binding of the complexes formed on either the NF-kappa B or NF-ELAM1 site. I...
bioRxiv (Cold Spring Harbor Laboratory), May 10, 2023
The sperm-specific channel CatSper (cation channel of sperm) controls the intracellular Ca 2+ con... more The sperm-specific channel CatSper (cation channel of sperm) controls the intracellular Ca 2+ concentration ([Ca 2+ ]i) and plays an essential role in sperm function. It is mainly activated by the steroid progesterone (P4) but is also promiscuously activated by a wide range of synthetic and physiological compounds. These compounds include diverse steroids whose action on the channel is so far still controversial. To investigate the effect of these compounds on CatSper and sperm function, we developed a high-throughput-screening (HTS) assay to measure changes in [Ca 2+ ]i in human sperm and screened 1,280 approved and off-patent drugs including 90 steroids from the Prestwick chemical library. More than half of the steroids tested (53%) induced an increase in [Ca 2+ ]i and reduced the P4-induced Ca 2+ influx in human sperm in a dose-dependent manner. Ten of the most potent steroids (activating and inhibiting) were selected for a detailed analysis of their action on CatSper and their ability to act on sperm motility, acrosomal exocytosis (AR), and penetration in viscous media. We found that these steroids show an inhibitory effect on P4 but not on prostaglandin E1-induced CatSper activation, suggesting that they compete for the same binding site as P4. Pregnenolone, dydrogesterone, epiandrosterone, nandrolone, and dehydroepiandrosterone acetate (DHEA) were found to activate CatSper at physiological concentrations. Stanozolol, epiandrosterone, and pregnenolone induced AR similarly to P4, whereas stanozolol and estropipate induced an increase in sperm penetration into viscous medium. Furthermore, using a hybrid approach integrating pharmacophore analysis and statistical modelling, we were able to screen in silico for steroids that can activate the channel and define the physicochemical and structural properties required for a steroid to exhibit agonist activity against CatSper. Overall, our results indicate that not only physiological but also synthetic steroids can modulate the activity of CatSper with varying potency and affect human sperm functions in vitro. .
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
The nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) multi-subunit complex is a highl... more The nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) multi-subunit complex is a highly abundant and central source of reactive oxygen species. NOX2 is a key enzyme of the innate immune system involved in antibacterial response, but excessive NOX2 activity is involved in oxidative stress and inflammation in many diseases. Inhibition of NOX2 has great potential as a therapeutic strategy. An intriguing pharmacological approach for inhibiting NOX2 is to target the p47phox subunit and thereby block the protein-protein interaction with p22phox, whereby assembling and activation of NOX2 is prevented. However, the shallow binding pocket of p47phox makes it difficult to develop drug-like p47phox/p22phox inhibitors. Recently, the small molecule LMH001 was reported to inhibit the p47phox/p22phox interaction, reduce endothelial NOX2 activity, and protect mice from angiotensin II-induced vascular oxidative stress. These noteworthy results could have significant impact on the field of NOX2 pharmacology, as specific and efficient inhibitors are scarce. Here, we synthesized and tested LMH001 to have it available as a positive control. We established a robust synthetic route for providing LMH001, but subsequently we experienced that LMH001 is chemically unstable in aqueous buffer. In addition, neither LMH001 nor its breakdown products were able to inhibit the p47phox/ p22phox interaction in a non-cellular fluorescence polarization assay. However, LHM001 was a weak inhibitor of NOX2 in a functional cell assay, but with same low potency as one of its breakdown products. These findings question the activity and suggested mechanism of LMH001 and constitute important information for other researchers interested in chemical probes for studying NOX2 biology.
The sperm-specific channel CatSper (cation channel ofsperm) controls the intracellular Ca2+concen... more The sperm-specific channel CatSper (cation channel ofsperm) controls the intracellular Ca2+concentration ([Ca2+]i) and plays an essential role in sperm function. It is mainly activated by the steroid progesterone (P4) but is also promiscuously activated by a wide range of synthetic and physiological compounds. These compounds include diverse steroids whose action on the channel is so far still controversial. To investigate the effect of these compounds on CatSper and sperm function, we developed a high-throughput-screening (HTS) assay to measure changes in [Ca2+]iin human sperm and screened 1,280 approved and off-patent drugs including 90 steroids from the Prestwick chemical library. More than half of the steroids tested (53%) induced an increase in [Ca2+]iand reduced the P4-induced Ca2+influx in human sperm in a dose-dependent manner. Ten of the most potent steroids (activating and inhibiting) were selected for a detailed analysis of their action on CatSper and their ability to act...
The nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) multi-subunit complex is a highl... more The nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) multi-subunit complex is a highly abundant and central source of reactive oxygen species. NOX2 is a key enzyme of the innate immune system involved in antibacterial response, but excessive NOX2 activity is involved in oxidative stress and inflammation in many diseases. Inhibition of NOX2 has great potential as a therapeutic strategy. An intriguing pharmacological approach for inhibiting NOX2 is to target the p47phox subunit and thereby block the protein-protein interaction with p22phox, whereby assembling and activation of NOX2 is prevented. However, the shallow binding pocket of p47phox makes it difficult to develop drug-like p47phox/p22phox inhibitors. Recently, the small molecule LMH001 was reported to inhibit the p47phox/p22phox interaction, reduce endothelial NOX2 activity, and protect mice from angiotensin II-induced vascular oxidative stress. These noteworthy results could have significant impact on the field of...
Hydrogen peroxide (H2O2) is a key defense component of host-microbe interaction. However, H2O2 co... more Hydrogen peroxide (H2O2) is a key defense component of host-microbe interaction. However, H2O2 concentrations generated by immune cells or epithelia are usually insufficient for bacterial killing and rather modulate bacterial responses. Here, we investigated the impact of sublethal H2O2 concentration on gene expression of E. coli BW25113 after 10 and 60 min of exposure. RNA-seq analysis revealed that approximately 12% of bacterial genes were strongly dysregulated 10 min following exposure to 2.5 mM H2O2. H2O2 exposure led to the activation of a specific antioxidant response and a general stress response. The latter was characterized by a transient down-regulation of genes involved in general metabolism, such as nucleic acid biosynthesis and translation, with a striking and coordinated down-regulation of genes involved in ribosome formation, and a sustained up-regulation of the SOS response. We confirmed the rapid transient and specific response mediated by the transcription factor O...
Background: The SARS-CoV-2 pandemic was particularly devastating for elderly people, and the unde... more Background: The SARS-CoV-2 pandemic was particularly devastating for elderly people, and the underlying mechanisms of the disease are still poorly understood. In this study, we investigated fusion inhibitory antibodies (fiAbs) in elderly and younger COVID-19 patients and analyzed predictive factors for their occurrence. Methods: Data and samples were collected in two cohorts of hospitalized patients. A fusion assay of SARS-CoV-2 spike-expressing cells with ACE2-expressing cells was used to quantify fiAbs in the serum of patients. Results: A total of 108 patients (52 elderly (mean age 85 ± 7 years); 56 young (mean age 52 ± 10 years)) were studied. The concentrations of fiAbs were lower in geriatric patients, as evidenced at high serum dilutions (1/512). The association between fiAbs and anti-Spike Ig levels was weak (correlation coefficient < 0.3), but statistically significant. Variables associated with fusion were the delay between the onset of symptoms and testing (HR = −2.69; ...
Several lines of evidence support the hypothesis that c-Jun N-terminal kinases (JNKs) play a crit... more Several lines of evidence support the hypothesis that c-Jun N-terminal kinases (JNKs) play a critical role in a wide range of disease states including cell death (apoptosis)-related and inflammatory disorders (epilepsy, brain, heart and renal ischemia, neurodegenerative diseases, multiple sclerosis, rheumatoid arthritis, and inflammatory bowel syndrome). The screening of a compound collection led to the identification of a 2-(benzoylaminomethyl)thiophene sulfonamide (AS004509, compound I) as a potent and selective JNK inhibitor. Chemistry and structure--activity relationship (SAR) studies performed around this novel kinase-inhibiting motif indicated that the left and central parts of the molecule were instrumental to maintaining potency at the enzyme. Accordingly, we investigated the JNK-inhibiting properties of a number of variants of the right-hand moiety of the molecule, which led to the identification of 2-(benzoylaminomethyl)thiophene sulfonamide benzotriazole (AS600292, compou...
Age-related hearing (ARHL) loss affects a large part of the human population with a major impact ... more Age-related hearing (ARHL) loss affects a large part of the human population with a major impact on our aging societies. Yet, underlying mechanisms are not understood, and no validated therapy or prevention exists. NADPH oxidases (NOX), are important sources of reactive oxygen species (ROS) in the cochlea and might therefore be involved in the pathogenesis of ARHL. Here we investigate ARHL in a mouse model. Wild type mice showed early loss of hearing and cochlear integrity, while animals deficient in the NOX subunit p22 phox remained unaffected up to six months. Genes of the excitatory pathway were down-regulated in p22 phox-deficient auditory neurons. Our results demonstrate that NOX activity leads to upregulation of genes of the excitatory pathway, to excitotoxic cochlear damage, and ultimately to ARHL. In the absence of functional NOXs, aging mice conserve hearing and cochlear morphology. Our study offers new insights into pathomechanisms and future therapeutic targets of ARHL.
Several lines of evidence support the hypothesis that c-Jun N-terminal kinases (JNKs) play a crit... more Several lines of evidence support the hypothesis that c-Jun N-terminal kinases (JNKs) play a critical role in a wide range of disease states including cell death (apoptosis)-related and inflammatory disorders (epilepsy, brain, heart and renal ischemia, neurodegenerative diseases, multiple sclerosis, rheumatoid arthritis, and inflammatory bowel syndrome). The screening of a compound collection led to the identification of a 2-(benzoylaminomethyl)thiophene sulfonamide (AS004509, compound I) as a potent and selective JNK inhibitor. Chemistry and structure--activity relationship (SAR) studies performed around this novel kinase-inhibiting motif indicated that the left and central parts of the molecule were instrumental to maintaining potency at the enzyme. Accordingly, we investigated the JNK-inhibiting properties of a number of variants of the right-hand moiety of the molecule, which led to the identification of 2-(benzoylaminomethyl)thiophene sulfonamide benzotriazole (AS600292, compou...
In the field of screening in general and especially in the kinase area, taking into consideration... more In the field of screening in general and especially in the kinase area, taking into consideration throughput and cost, fluorescence-and luminescence-based assays have been developed as alternatives to radioactivity-based assays. However, fluorescence-based technologies are not devoid of pitfalls. One of the main problems is interference from autofluorescent compounds and the incidence of false-positives as exemplified here with a fluorescence polarization (FP)-based assay. Using the scintillation proximity assay as the in-house standard, we assessed several alternatives to radioactive methods, namely, the amplified luminescent proximity homeogeneous assay screen (ALPHAScreen™, Perkin-Elmer Life Sciences, Boston, MA), enzyme fragment complementation, FP, and nanofluidics-based fluorescence intensity. Data comparing the sensitivity, robustness, relative sensitivity to autofluorescent compounds, enzyme consumption, and relative costs of each assay for one common kinase are presented. Results obtained seem to favor the nanofluidics mobility shift assay as a method of choice, followed by the direct FP approach, using generic high-molecularweight phosphate group-binders.
Phenazopyridine is a widely used drug against urinary tract pain. The compound has also been show... more Phenazopyridine is a widely used drug against urinary tract pain. The compound has also been shown to enhance neural differentiation of pluripotent stem cells. However, its mechanism of action is not understood. Based on its chemical structure, we hypothesized that phenazopyridine could be a kinase inhibitor. Phenazopyridine was investigated in the following experimental systems: 1) activity of kinases in pluripotent stem cells; 2) binding to recombinant kinases, and 3) functional impact on pluripotent stem cells. Upon addition to pluripotent stem cells, phenazopyridine induced changes in kinase activities, particularly involving Mitogen-Activated Protein Kinases, Cyclin-Dependent Kinases, and AKT pathway kinases. To identify the primary targets of phenazopyridine, we screened its interactions with 401 human kinases. Dose-inhibition curves showed that three of these kinases interacted with phenazopyridine with sub-micromolar binding affinities: cyclin-G-associated kinase, and the tw...
Cytokines induce the expression of E-selectin, VCAM-1, and ICAM-1 on human umbilical vein endothe... more Cytokines induce the expression of E-selectin, VCAM-1, and ICAM-1 on human umbilical vein endothelial cells (HUVECs). We show that expression of these surface proteins is differently affected by cAMP. Increased cAMP levels decrease E-selectin and VCAM-1 but increase ICAM-1 expression. We demonstrate by mRNA half-life analysis and nuclear run-on assays that the cAMP repression of E-selectin occurs at the transcription level. This effect is abolished by protein kinase A inhibition, suggesting that repression is mediated by protein kinase A-driven phosphorylation. We found that a minimal E-selectin promoter sequence necessary to confer cytokine inducibility is also sufficient to mimic the cAMP effect in transfected HUVECs. Previously we characterized two regions (NF-kappa B and NF-ELAM1) of the minimal promoter that bind transcription factors necessary for E-selectin induction, Increased cAMP did not alter the binding of the complexes formed on either the NF-kappa B or NF-ELAM1 site. I...
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