This document describes the identification of potent phosphodiesterase (PDE) inhibitors that demonstrate cyclic nucleotide-dependent functions in apicomplexan parasites. The most potent inhibitor identified was 5-Benzyl-3-isopropyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (BIPPO), which potently inhibited recombinant P. falciparum Pf PDEα and induced protein kinase G (PKG)-dependent egress in T. gondii and P. falciparum by promoting microneme secretion. BIPPO also promoted cAMP-dependent phosphorylation of a P. falciparum ligand critical for host cell invasion,
This document summarizes a study investigating the role of a Plasmodium falciparum S33 proline aminopeptidase (PfPAP) in changes to host red blood cell deformability. The key findings are:
1) PfPAP contains a predicted protein export element suggesting it is exported into infected red blood cells. In silico modeling and recombinant protein studies confirmed PfPAP is a proline aminopeptidase.
2) Genetic deletion of PfPAP in P. falciparum led to an increase in the deformability of infected red blood cells and reduced adherence of infected cells to the endothelial cell receptor CD36 under flow conditions.
3) These results suggest PfP
The document summarizes a study that identified potent and selective inhibitors of the Plasmodium falciparum M18 aspartyl aminopeptidase (PfM18AAP) enzyme via high-throughput screening. PfM18AAP plays an important role in malaria parasite growth and is a potential drug target. A fluorescence-based assay was developed to screen over 292,000 compounds, identifying two structurally related compounds that potently and selectively inhibited PfM18AAP in the low micromolar range. Both compounds were found to be noncompetitive inhibitors of PfM18AAP and inhibited malaria parasite growth, demonstrating their potential as antimalarial therapies.
J. biol. chem. 2016-shao-jbc.m116.724401andrei andrei
FBXO3 promotes ubiquitylation and transcriptional activity of AIRE. The study found that the E3 ubiquitin ligase FBXO3 interacts with phosphorylated residues on AIRE and promotes its ubiquitylation. This post-translational modification increases the interaction between AIRE and P-TEFb, potentiating their transcriptional activity on tissue-specific antigen genes in the thymus. Knockdown of FBXO3 decreased ubiquitylation and transcriptional activity of AIRE.
This document summarizes a study that created an inducible mouse model expressing pathological human tau (PH-Tau) to investigate the effects of tau phosphorylation levels on cognition and neurodegeneration. The study found that even low levels of PH-Tau expression (4% of total tau) led to cognitive deficits through synaptic dysfunction, while higher levels (14% of total tau) induced neuronal death and further cognitive impairment. This suggests that abnormal tau can cause cognitive impairment through two different mechanisms depending on its expression level - synaptic dysfunction at low levels and neuronal loss at high levels.
Poster prepared by James Nyagwange, Nicola Ternette, Edwin Tijhaar, Roger Pelle and Vish Nene for the Keystone Symposium on New Approaches to Vaccines for Human and Veterinary Tropical Diseases, Cape Town, 22-26 May 2016
Immunogenicity and protection of the Theileria parva CTL antigen Tp1, with o...ILRI
Poster prepared by N. Svitek, R. Saya, E. Awino, S. Gilbert, J. Poole, V. Nene and L. Steinaa for the Keystone Symposium on New Approaches to Vaccines for Human and Veterinary Tropical Diseases, Cape Town, 22-26 May 2016
Discovery of novel CTL epitopes by peptide library screening of CTL lines fro...ILRI
Poster prepared by N. Svitek, R. Saya, E. Awino, M. Nielsen, N. MacHugh, J.C. da Silva, V. Nene and L. Steinaa for the Keystone Symposium on New Approaches to Vaccines for Human and Veterinary Tropical Diseases, Cape Town, 22-26 May 2016
This study investigated DNA damage signaling during Epstein-Barr virus (EBV) lytic replication. The key findings were:
1) Markers of DNA damage signaling like phosphorylated ATM (pATM) and 53BP1 were induced upon expression of the EBV lytic transactivator ZEBRA, both in the presence and absence of EBV DNA replication.
2) pATM foci formed in response to ZEBRA expression in the majority of lytically reactivated cells, indicating DNA damage signaling is activated early in the lytic cycle before replication.
3) pATM and 53BP1 foci were observed in cells expressing early lytic proteins EA-
This document summarizes a study investigating the role of a Plasmodium falciparum S33 proline aminopeptidase (PfPAP) in changes to host red blood cell deformability. The key findings are:
1) PfPAP contains a predicted protein export element suggesting it is exported into infected red blood cells. In silico modeling and recombinant protein studies confirmed PfPAP is a proline aminopeptidase.
2) Genetic deletion of PfPAP in P. falciparum led to an increase in the deformability of infected red blood cells and reduced adherence of infected cells to the endothelial cell receptor CD36 under flow conditions.
3) These results suggest PfP
The document summarizes a study that identified potent and selective inhibitors of the Plasmodium falciparum M18 aspartyl aminopeptidase (PfM18AAP) enzyme via high-throughput screening. PfM18AAP plays an important role in malaria parasite growth and is a potential drug target. A fluorescence-based assay was developed to screen over 292,000 compounds, identifying two structurally related compounds that potently and selectively inhibited PfM18AAP in the low micromolar range. Both compounds were found to be noncompetitive inhibitors of PfM18AAP and inhibited malaria parasite growth, demonstrating their potential as antimalarial therapies.
J. biol. chem. 2016-shao-jbc.m116.724401andrei andrei
FBXO3 promotes ubiquitylation and transcriptional activity of AIRE. The study found that the E3 ubiquitin ligase FBXO3 interacts with phosphorylated residues on AIRE and promotes its ubiquitylation. This post-translational modification increases the interaction between AIRE and P-TEFb, potentiating their transcriptional activity on tissue-specific antigen genes in the thymus. Knockdown of FBXO3 decreased ubiquitylation and transcriptional activity of AIRE.
This document summarizes a study that created an inducible mouse model expressing pathological human tau (PH-Tau) to investigate the effects of tau phosphorylation levels on cognition and neurodegeneration. The study found that even low levels of PH-Tau expression (4% of total tau) led to cognitive deficits through synaptic dysfunction, while higher levels (14% of total tau) induced neuronal death and further cognitive impairment. This suggests that abnormal tau can cause cognitive impairment through two different mechanisms depending on its expression level - synaptic dysfunction at low levels and neuronal loss at high levels.
Poster prepared by James Nyagwange, Nicola Ternette, Edwin Tijhaar, Roger Pelle and Vish Nene for the Keystone Symposium on New Approaches to Vaccines for Human and Veterinary Tropical Diseases, Cape Town, 22-26 May 2016
Immunogenicity and protection of the Theileria parva CTL antigen Tp1, with o...ILRI
Poster prepared by N. Svitek, R. Saya, E. Awino, S. Gilbert, J. Poole, V. Nene and L. Steinaa for the Keystone Symposium on New Approaches to Vaccines for Human and Veterinary Tropical Diseases, Cape Town, 22-26 May 2016
Discovery of novel CTL epitopes by peptide library screening of CTL lines fro...ILRI
Poster prepared by N. Svitek, R. Saya, E. Awino, M. Nielsen, N. MacHugh, J.C. da Silva, V. Nene and L. Steinaa for the Keystone Symposium on New Approaches to Vaccines for Human and Veterinary Tropical Diseases, Cape Town, 22-26 May 2016
This study investigated DNA damage signaling during Epstein-Barr virus (EBV) lytic replication. The key findings were:
1) Markers of DNA damage signaling like phosphorylated ATM (pATM) and 53BP1 were induced upon expression of the EBV lytic transactivator ZEBRA, both in the presence and absence of EBV DNA replication.
2) pATM foci formed in response to ZEBRA expression in the majority of lytically reactivated cells, indicating DNA damage signaling is activated early in the lytic cycle before replication.
3) pATM and 53BP1 foci were observed in cells expressing early lytic proteins EA-
Cloning and extracellular expression of recombinant tissue plasminogen activa...bioejjournal
This document summarizes research conducted to clone and express recombinant tissue plasminogen activator (rt-PA) using the methylotrophic yeast Pichia pastoris. Specifically, the tissue plasminogen activator gene was cloned into the pICZαA expression vector. This construct was then transformed into Pichia pastoris X33 cells. One transformant, named Pichia tPA-3, showed expression of rt-PA at 66 kDa on SDS-PAGE. Size exclusion chromatography also showed a product peak at the same retention time as a reference tPA standard. Thus, the researchers were able to successfully clone and express rt-PA in the Pichia pastoris system.
Cloning and extracellular expression of recombinant tissue plasminogen activa...bioejjournal
Tissue plasminogen activator (tPA) has noteworthy application in treatment of acute myocardial
infarctions. This study focuses on expression of rt-PA using microbial systems in order to reduce cost
without compromising on quality as an alternative to commercial (rt-PA)produced by using mammalian
host systems. In the present study, Pichia pastoris X-33strain was used as a host with pICZαA expression
vector to obtain extracellular expression of full length tPA gene. Specific primers were designed in such a
way to get native tPA protein sequence in subsequent purification steps. Further, construct pICZαA-tPA
was developed and electroporated into host to achieve stablert-PA gene by achieving genome integration. The transformants were screened for phenotypic characters.Mut+
phenotypic colony named Pichia tPA-3
showed expression of rt-PA at 66 kDa on SDS PAGE. Size Exclusion Chromatography (SEC) was
performed, resulting in product peak at same RT as reference standard. (alteplase).Cloning and expression
of rt-PA was successfully achieved in microbial system. Further process optimization at larger scales will surely provide cost effective alternative to mammalian system forrt-PA production
Cloning and Extracellular Expression of Recombinant Tissue Plasminogen Activa...bioejjournal
This document summarizes research conducted to clone and express recombinant tissue plasminogen activator (rt-PA) using the methylotrophic yeast Pichia pastoris. Specifically, the full-length tPA gene was amplified by PCR and cloned into the pICZαA expression vector. This construct was then transformed into Pichia pastoris X33 cells. One transformant, named Pichia tPA-3, showed extracellular expression of rt-PA at 66 kDa on SDS-PAGE after induction with methanol over 144 hours. Size exclusion chromatography of samples from this transformant showed a product peak at the same retention time as a reference tPA standard. Thus, the researchers successfully achieved cloning and extracellular expression of
This document summarizes a study examining the role of the long non-coding RNA Saf and splicing factor 45 in regulating alternative splicing of the Fas pre-mRNA and production of soluble Fas protein. The main findings are:
1) Saf is predominantly localized to the nucleus of cells. Overexpression of Saf in cell lines resulted in minimal effects on global gene expression.
2) Saf interacts with Fas pre-mRNA and splicing factor 45. This interaction promotes the skipping of exon 6 in the Fas pre-mRNA, leading to increased production of soluble Fas protein.
3) Soluble Fas protein protects cells from Fas ligand-induced apoptosis by binding to the ligand and blocking
This document summarizes a study investigating the roles of Rho and ADP-ribosylation Factor (ARF) GTPases in regulating phospholipase D (PLD) activity in human lung adenocarcinoma cells. The study used recombinant Sindbis viruses to express Clostridium botulinum C3 exoenzyme (which inactivates Rho) and a dominant-negative Rho mutant in the cells. Expression of C3 or the mutant Rho increased basal PLD activity and decreased phorbol ester-stimulated PLD activity. Bradykinin- or sphingosine-stimulated PLD activity, which had additive effects, was abolished by C3 or mutant Rho expression. Brefeld
Polymorphism affecting drug metabolism .heenakazi4
This document discusses genetic polymorphisms that affect drug metabolism. It begins by defining genetic polymorphism and describing the main types, including single nucleotide polymorphisms, insertions and deletions, and nucleotide repeat polymorphisms. It then discusses how genetic polymorphisms can alter drug metabolism through variations in metabolic enzyme activity and discusses specific examples of polymorphisms in key drug metabolizing enzymes like CYP2D6, CYP2C9, and N-acetyltransferases. The document emphasizes how understanding genetic variations in drug metabolism is important for predicting inter-individual differences in drug responses.
1) Lipopolysaccharide (LPS) causes apoptotic cell death of immature CD4+ CD8+ thymocytes, which is linked to immune dysfunction during sepsis. 2) This study investigated whether enhanced generation of thromboxane A2 (TXA2) plays a role in LPS-induced thymocyte apoptosis. 3) The results indicate that TXA2 mediates a portion of apoptotic thymocyte death caused by LPS, as thymocyte apoptosis was attenuated in mice lacking TXA2 receptors, though global inhibition of prostaglandin synthesis did not affect cell death.
Genetic polymorphisms can affect how individuals metabolize and respond to drugs. The document discusses how single nucleotide polymorphisms (SNPs) in genes encoding drug-metabolizing enzymes like the cytochrome P450 system can result in poor, intermediate, normal, extensive, or ultra-rapid metabolizers. This genetic variation impacts the metabolism of many drugs and can influence their effects as well as drug interactions. The cytochrome P450 2C19 enzyme, which is important in metabolizing diazepam, shows polymorphisms that lead to different drug responses and side effects between ethnic populations. Understanding these pharmacogenomic factors is important for optimizing drug therapy.
Cell signaling her2 expression in breast cancerOmid Yeganeh
This document discusses cell signaling and HER2 expression in breast cancer. It provides information on:
1) Cell signaling can be mechanical or biochemical based on the type of signal transmitted between cells. Biochemical signals include proteins, lipids, ions and gases.
2) Communication between cells controls growth, differentiation and metabolic processes through direct contact or over short and long distances using signaling molecules called hormones.
3) The HER2 signaling pathway involves tyrosine kinase receptors that activate downstream signaling cascades upon ligand binding, influencing processes like proliferation and survival. Overexpression of HER2 occurs in 20-30% of breast cancers.
4) The PI3K-Akt pathway is an important signaling cascade downstream of HER
Venters Molecular and Cellular Biology 2011 2253-2261Jordan Irvin
This study investigates the contributions of conserved regions of the essential yeast protein Mot1 to genome-wide gene regulation and transcription factor (TBP) recruitment. The researchers used a transient replacement strategy to delete conserved regions of Mot1 and analyzed the effects on gene expression and TBP occupancy. They found that the four conserved regions of Mot1 are generally required for all Mot1-regulated genes. Deletion of the ATPase region specifically altered transcriptional dependence for some genes and caused TBP to redistribute away from Mot1-inhibited genes and to other normally Mot1-independent genes. This suggests that Mot1 uses ATP hydrolysis to redistribute accessible TBP between sites of high and low intrinsic preference.
Toll-like receptors (TLRs) are immune sensors that detect pathogen-associated molecular patterns and induce inflammatory responses. TLRs activate signaling cascades that lead to the production of cytokines and immune cell activation through adaptor proteins and kinases. Dysregulation of TLR signaling can cause or contribute to immune deficiencies, autoimmune diseases, and cancers. The document discusses TLR structure and function, downstream signaling pathways, regulation of the response, and diseases associated with TLR signaling abnormalities.
This study investigated how oxidative stress activates the PI3K pathway in neurons affected by neurodegenerative diseases. The researchers found that ingestion of the oxidative stress inducer Paraquat in Drosophila larvae caused axonal transport defects and neuronal cell death. Expressing active PI3K suppressed Paraquat-mediated cell death but not axonal blocks, indicating PI3K acts downstream of transport defects. Expression of active PI3K also suppressed cell death from polyQ protein expression but did not affect associated transport defects. Dominant negative PI3K disrupted normal transport of huntingtin protein, linking PI3K directly to transport. Together, the findings suggest axonal transport defects activate the PI3K pathway to decrease oxidative stress-induced
This document discusses methods for identifying protein-protein interactions, including the yeast two-hybrid system and co-immunoprecipitation. It provides details on how each technique works and describes an example application investigating interactions in B cell receptor signaling pathways. Key proteins in the B cell receptor pathway are identified that could be used as baits in future experiments.
This study uses real-time PCR arrays to analyze gene expression profiles in liver cells treated with different diabetes drugs to better understand their toxicity mechanisms. The gene expression profile of cells treated with the withdrawn drug Rezulin (Troglitazone) was found to be distinct from the profiles of cells treated with the safer drugs Avandia (Rosiglitazone) and Actos (Pioglitazone). In particular, two genes were found to have much higher expression levels in cells treated with Rezulin compared to the other drugs, indicating these genes may help explain Rezulin's idiosyncratic liver toxicity. The gene expression profiling approach was able to differentiate the effects of different drugs and holds promise for predicting
Investigating the function of a novel protein from Anoectochilus formosanus w...Cây thuốc Việt
Anoectochilus formosanus is a therapeutic orchid appreciated as a traditional Chinese medicine in Asia. The extracts of A. formosanus have been reported to possess hepatoprotective, anti-inflammatory, and anti-tumor activates. A novel protein was isolated from A. formosanus, and its immunomodulatory effect on murine peritoneal macrophage was investigated. Macrophages obtained from ascites of thioglycollate-induced BALB/c were co-cultured with IPAF (0–20μg/ml) for 24h and then harvested for flow cytometry analysis. The cytokine/chemokine production was measured by real time PCR and ELISA. The interaction between IPA and toll like receptors (TLRs) was investigated by TLR gene knockout (KO) mice and fluorescence labeled IPAF. The activation of NF-κB was assessed by EMSA. IPAF stimulated the TNF-α and IL-1β production, upregulated the CD86 and MHC II expression, and enhanced the phagocytic activity of macrophages. IPAF induced gene expression of IL-12 and Th1-assosiated cytokines/chemokines. The stimulating effect of IPAF was
impaired, and the IPAF–macrophage interaction was reduced in TLR4−/− C57BL/10ScNJ mice. In addition, IPAF stimulated expressions of TLR signal-related genes and the activation of NF-κB. IPAF could induce classical activated macrophage differentiation via TLR4-dependent NF-κB activation and had potential of IPAF to modulate the Th1 response. These findings provided valuable information regarding the immune modulatory mechanism of A. formosanus, and indicated the possibility of IPAF as a potential peptide drug
Yeast 2 hybrid system ppt by meera qaiserQaiser Sethi
The yeast two-hybrid system is a technique used to study protein-protein interactions. It involves creating two fusion proteins, one with a DNA-binding domain and one with an activation domain. If the proteins of interest interact, they will reconstitute a functional transcription factor that activates a reporter gene. This allows researchers to identify novel interactions and characterize known interactions, helping to further understand biological processes at the molecular level.
Stable transfected HEK293 cells expressing various drug transporters like OATP1A2, OATP1B1, OATP1B3, and OATP2B1 were generated. The transporter expression was confirmed and the cells were characterized by determining transport of specific substrates and inhibition by known inhibitors. Uptake assays found the cells actively transported representative substrates for each transporter and this uptake was inhibited by known inhibitors, demonstrating the functional expression of each transporter in the respective cell lines.
International Journal of Pharmaceutical Science Invention (IJPSI) is an international journal intended for professionals and researchers in all fields of Pahrmaceutical Science. IJPSI publishes research articles and reviews within the whole field Pharmacy and Pharmaceutical Science, new teaching methods, assessment, validation and the impact of new technologies and it will continue to provide information on the latest trends and developments in this ever-expanding subject. The publications of papers are selected through double peer reviewed to ensure originality, relevance, and readability. The articles published in our journal can be accessed online.
Expression, purification and spectroscopic characterization of the cytochrome...John Clarkson
K.J. McLean, M.R. Cheesman, S.L. Rivers, A. Richmond, D. Leys, S.K. Chapman, G.A. Reid, N.C. Price, S.M. Kelly, J. Clarkson, W.E Smith & A.W. Munro, “Expression, Purification and Spectroscopic Characterization of the Cytochrome P450 CYP121 from Mycobacterium Tuberculosis”, J. Inorganic Biochemistry, 91, 527-541, 2002.
El documento describe cómo la biología humana está orientada a satisfacer las necesidades energéticas del músculo y cómo nuestra adaptación actual al entorno difiere de aquella para la que evolucionamos. Explica que la contracción muscular activa la proteína AMPK, la cual regula el metabolismo de la glucosa y los ácidos grasos de forma independiente a la insulina. También describe cómo la activación de AMPK mejora la sensibilidad a la insulina y el consumo de glucosa en el músculo, lo que tiene implicaciones positivas para la preven
Cloning and extracellular expression of recombinant tissue plasminogen activa...bioejjournal
This document summarizes research conducted to clone and express recombinant tissue plasminogen activator (rt-PA) using the methylotrophic yeast Pichia pastoris. Specifically, the tissue plasminogen activator gene was cloned into the pICZαA expression vector. This construct was then transformed into Pichia pastoris X33 cells. One transformant, named Pichia tPA-3, showed expression of rt-PA at 66 kDa on SDS-PAGE. Size exclusion chromatography also showed a product peak at the same retention time as a reference tPA standard. Thus, the researchers were able to successfully clone and express rt-PA in the Pichia pastoris system.
Cloning and extracellular expression of recombinant tissue plasminogen activa...bioejjournal
Tissue plasminogen activator (tPA) has noteworthy application in treatment of acute myocardial
infarctions. This study focuses on expression of rt-PA using microbial systems in order to reduce cost
without compromising on quality as an alternative to commercial (rt-PA)produced by using mammalian
host systems. In the present study, Pichia pastoris X-33strain was used as a host with pICZαA expression
vector to obtain extracellular expression of full length tPA gene. Specific primers were designed in such a
way to get native tPA protein sequence in subsequent purification steps. Further, construct pICZαA-tPA
was developed and electroporated into host to achieve stablert-PA gene by achieving genome integration. The transformants were screened for phenotypic characters.Mut+
phenotypic colony named Pichia tPA-3
showed expression of rt-PA at 66 kDa on SDS PAGE. Size Exclusion Chromatography (SEC) was
performed, resulting in product peak at same RT as reference standard. (alteplase).Cloning and expression
of rt-PA was successfully achieved in microbial system. Further process optimization at larger scales will surely provide cost effective alternative to mammalian system forrt-PA production
Cloning and Extracellular Expression of Recombinant Tissue Plasminogen Activa...bioejjournal
This document summarizes research conducted to clone and express recombinant tissue plasminogen activator (rt-PA) using the methylotrophic yeast Pichia pastoris. Specifically, the full-length tPA gene was amplified by PCR and cloned into the pICZαA expression vector. This construct was then transformed into Pichia pastoris X33 cells. One transformant, named Pichia tPA-3, showed extracellular expression of rt-PA at 66 kDa on SDS-PAGE after induction with methanol over 144 hours. Size exclusion chromatography of samples from this transformant showed a product peak at the same retention time as a reference tPA standard. Thus, the researchers successfully achieved cloning and extracellular expression of
This document summarizes a study examining the role of the long non-coding RNA Saf and splicing factor 45 in regulating alternative splicing of the Fas pre-mRNA and production of soluble Fas protein. The main findings are:
1) Saf is predominantly localized to the nucleus of cells. Overexpression of Saf in cell lines resulted in minimal effects on global gene expression.
2) Saf interacts with Fas pre-mRNA and splicing factor 45. This interaction promotes the skipping of exon 6 in the Fas pre-mRNA, leading to increased production of soluble Fas protein.
3) Soluble Fas protein protects cells from Fas ligand-induced apoptosis by binding to the ligand and blocking
This document summarizes a study investigating the roles of Rho and ADP-ribosylation Factor (ARF) GTPases in regulating phospholipase D (PLD) activity in human lung adenocarcinoma cells. The study used recombinant Sindbis viruses to express Clostridium botulinum C3 exoenzyme (which inactivates Rho) and a dominant-negative Rho mutant in the cells. Expression of C3 or the mutant Rho increased basal PLD activity and decreased phorbol ester-stimulated PLD activity. Bradykinin- or sphingosine-stimulated PLD activity, which had additive effects, was abolished by C3 or mutant Rho expression. Brefeld
Polymorphism affecting drug metabolism .heenakazi4
This document discusses genetic polymorphisms that affect drug metabolism. It begins by defining genetic polymorphism and describing the main types, including single nucleotide polymorphisms, insertions and deletions, and nucleotide repeat polymorphisms. It then discusses how genetic polymorphisms can alter drug metabolism through variations in metabolic enzyme activity and discusses specific examples of polymorphisms in key drug metabolizing enzymes like CYP2D6, CYP2C9, and N-acetyltransferases. The document emphasizes how understanding genetic variations in drug metabolism is important for predicting inter-individual differences in drug responses.
1) Lipopolysaccharide (LPS) causes apoptotic cell death of immature CD4+ CD8+ thymocytes, which is linked to immune dysfunction during sepsis. 2) This study investigated whether enhanced generation of thromboxane A2 (TXA2) plays a role in LPS-induced thymocyte apoptosis. 3) The results indicate that TXA2 mediates a portion of apoptotic thymocyte death caused by LPS, as thymocyte apoptosis was attenuated in mice lacking TXA2 receptors, though global inhibition of prostaglandin synthesis did not affect cell death.
Genetic polymorphisms can affect how individuals metabolize and respond to drugs. The document discusses how single nucleotide polymorphisms (SNPs) in genes encoding drug-metabolizing enzymes like the cytochrome P450 system can result in poor, intermediate, normal, extensive, or ultra-rapid metabolizers. This genetic variation impacts the metabolism of many drugs and can influence their effects as well as drug interactions. The cytochrome P450 2C19 enzyme, which is important in metabolizing diazepam, shows polymorphisms that lead to different drug responses and side effects between ethnic populations. Understanding these pharmacogenomic factors is important for optimizing drug therapy.
Cell signaling her2 expression in breast cancerOmid Yeganeh
This document discusses cell signaling and HER2 expression in breast cancer. It provides information on:
1) Cell signaling can be mechanical or biochemical based on the type of signal transmitted between cells. Biochemical signals include proteins, lipids, ions and gases.
2) Communication between cells controls growth, differentiation and metabolic processes through direct contact or over short and long distances using signaling molecules called hormones.
3) The HER2 signaling pathway involves tyrosine kinase receptors that activate downstream signaling cascades upon ligand binding, influencing processes like proliferation and survival. Overexpression of HER2 occurs in 20-30% of breast cancers.
4) The PI3K-Akt pathway is an important signaling cascade downstream of HER
Venters Molecular and Cellular Biology 2011 2253-2261Jordan Irvin
This study investigates the contributions of conserved regions of the essential yeast protein Mot1 to genome-wide gene regulation and transcription factor (TBP) recruitment. The researchers used a transient replacement strategy to delete conserved regions of Mot1 and analyzed the effects on gene expression and TBP occupancy. They found that the four conserved regions of Mot1 are generally required for all Mot1-regulated genes. Deletion of the ATPase region specifically altered transcriptional dependence for some genes and caused TBP to redistribute away from Mot1-inhibited genes and to other normally Mot1-independent genes. This suggests that Mot1 uses ATP hydrolysis to redistribute accessible TBP between sites of high and low intrinsic preference.
Toll-like receptors (TLRs) are immune sensors that detect pathogen-associated molecular patterns and induce inflammatory responses. TLRs activate signaling cascades that lead to the production of cytokines and immune cell activation through adaptor proteins and kinases. Dysregulation of TLR signaling can cause or contribute to immune deficiencies, autoimmune diseases, and cancers. The document discusses TLR structure and function, downstream signaling pathways, regulation of the response, and diseases associated with TLR signaling abnormalities.
This study investigated how oxidative stress activates the PI3K pathway in neurons affected by neurodegenerative diseases. The researchers found that ingestion of the oxidative stress inducer Paraquat in Drosophila larvae caused axonal transport defects and neuronal cell death. Expressing active PI3K suppressed Paraquat-mediated cell death but not axonal blocks, indicating PI3K acts downstream of transport defects. Expression of active PI3K also suppressed cell death from polyQ protein expression but did not affect associated transport defects. Dominant negative PI3K disrupted normal transport of huntingtin protein, linking PI3K directly to transport. Together, the findings suggest axonal transport defects activate the PI3K pathway to decrease oxidative stress-induced
This document discusses methods for identifying protein-protein interactions, including the yeast two-hybrid system and co-immunoprecipitation. It provides details on how each technique works and describes an example application investigating interactions in B cell receptor signaling pathways. Key proteins in the B cell receptor pathway are identified that could be used as baits in future experiments.
This study uses real-time PCR arrays to analyze gene expression profiles in liver cells treated with different diabetes drugs to better understand their toxicity mechanisms. The gene expression profile of cells treated with the withdrawn drug Rezulin (Troglitazone) was found to be distinct from the profiles of cells treated with the safer drugs Avandia (Rosiglitazone) and Actos (Pioglitazone). In particular, two genes were found to have much higher expression levels in cells treated with Rezulin compared to the other drugs, indicating these genes may help explain Rezulin's idiosyncratic liver toxicity. The gene expression profiling approach was able to differentiate the effects of different drugs and holds promise for predicting
Investigating the function of a novel protein from Anoectochilus formosanus w...Cây thuốc Việt
Anoectochilus formosanus is a therapeutic orchid appreciated as a traditional Chinese medicine in Asia. The extracts of A. formosanus have been reported to possess hepatoprotective, anti-inflammatory, and anti-tumor activates. A novel protein was isolated from A. formosanus, and its immunomodulatory effect on murine peritoneal macrophage was investigated. Macrophages obtained from ascites of thioglycollate-induced BALB/c were co-cultured with IPAF (0–20μg/ml) for 24h and then harvested for flow cytometry analysis. The cytokine/chemokine production was measured by real time PCR and ELISA. The interaction between IPA and toll like receptors (TLRs) was investigated by TLR gene knockout (KO) mice and fluorescence labeled IPAF. The activation of NF-κB was assessed by EMSA. IPAF stimulated the TNF-α and IL-1β production, upregulated the CD86 and MHC II expression, and enhanced the phagocytic activity of macrophages. IPAF induced gene expression of IL-12 and Th1-assosiated cytokines/chemokines. The stimulating effect of IPAF was
impaired, and the IPAF–macrophage interaction was reduced in TLR4−/− C57BL/10ScNJ mice. In addition, IPAF stimulated expressions of TLR signal-related genes and the activation of NF-κB. IPAF could induce classical activated macrophage differentiation via TLR4-dependent NF-κB activation and had potential of IPAF to modulate the Th1 response. These findings provided valuable information regarding the immune modulatory mechanism of A. formosanus, and indicated the possibility of IPAF as a potential peptide drug
Yeast 2 hybrid system ppt by meera qaiserQaiser Sethi
The yeast two-hybrid system is a technique used to study protein-protein interactions. It involves creating two fusion proteins, one with a DNA-binding domain and one with an activation domain. If the proteins of interest interact, they will reconstitute a functional transcription factor that activates a reporter gene. This allows researchers to identify novel interactions and characterize known interactions, helping to further understand biological processes at the molecular level.
Stable transfected HEK293 cells expressing various drug transporters like OATP1A2, OATP1B1, OATP1B3, and OATP2B1 were generated. The transporter expression was confirmed and the cells were characterized by determining transport of specific substrates and inhibition by known inhibitors. Uptake assays found the cells actively transported representative substrates for each transporter and this uptake was inhibited by known inhibitors, demonstrating the functional expression of each transporter in the respective cell lines.
International Journal of Pharmaceutical Science Invention (IJPSI) is an international journal intended for professionals and researchers in all fields of Pahrmaceutical Science. IJPSI publishes research articles and reviews within the whole field Pharmacy and Pharmaceutical Science, new teaching methods, assessment, validation and the impact of new technologies and it will continue to provide information on the latest trends and developments in this ever-expanding subject. The publications of papers are selected through double peer reviewed to ensure originality, relevance, and readability. The articles published in our journal can be accessed online.
Expression, purification and spectroscopic characterization of the cytochrome...John Clarkson
K.J. McLean, M.R. Cheesman, S.L. Rivers, A. Richmond, D. Leys, S.K. Chapman, G.A. Reid, N.C. Price, S.M. Kelly, J. Clarkson, W.E Smith & A.W. Munro, “Expression, Purification and Spectroscopic Characterization of the Cytochrome P450 CYP121 from Mycobacterium Tuberculosis”, J. Inorganic Biochemistry, 91, 527-541, 2002.
El documento describe cómo la biología humana está orientada a satisfacer las necesidades energéticas del músculo y cómo nuestra adaptación actual al entorno difiere de aquella para la que evolucionamos. Explica que la contracción muscular activa la proteína AMPK, la cual regula el metabolismo de la glucosa y los ácidos grasos de forma independiente a la insulina. También describe cómo la activación de AMPK mejora la sensibilidad a la insulina y el consumo de glucosa en el músculo, lo que tiene implicaciones positivas para la preven
The forth lecture about the "Cell".
Here, I am discussing the several signaling pathways.....It is highly dependent on the 3rd lecture; Receptors.
Enjoy :)
AMP-activated protein kinase (AMPK) is an enzyme that acts as a cellular energy sensor. It is activated by increases in the AMP:ATP ratio caused by metabolic stresses that deplete ATP. When activated, AMPK works to switch on catabolic pathways that generate ATP while switching off ATP-consuming processes like biosynthesis. AMPK activation improves blood glucose and lipid levels, making it a promising target for treating diabetes and other metabolic disorders. AMPK regulates glucose and lipid metabolism in the liver, skeletal muscle, pancreas and hypothalamus.
This document discusses phosphodiesterase inhibitors, which were first isolated from rat brain in 1972 and inhibit the breakdown of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). It describes several subtypes of phosphodiesterase inhibitors, including non-selective inhibitors like caffeine and theophylline, as well as selective inhibitors of PDE1-5 that are used to treat conditions like erectile dysfunction, pulmonary arterial hypertension, chronic obstructive pulmonary disease, and intermittent claudication.
This document provides an overview of protein kinase cascades. It begins with an introduction to protein kinases and their function in phosphorylating proteins and playing roles in cellular processes. It then discusses the classification of protein kinases based on the amino acid they phosphorylate. Next, it explains the basic mechanism of phosphorylation by protein kinases using ATP. It proceeds to describe protein kinase cascades where one kinase phosphorylates and activates the next in a chain. Examples of double phosphorylation and multi-layer perceptrons in cascades are illustrated. Finally, some biological examples of different types of protein kinases are mentioned before concluding with references.
Phosphodiesterase inhibitors work by preventing the breakdown of cyclic nucleotides like cAMP and cGMP. There are 12 families of PDE enzymes that are involved in breaking down these second messenger molecules. Selective PDE inhibitors have been developed that target specific PDE families for conditions like erectile dysfunction, pulmonary hypertension, heart failure, and asthma. Future research is exploring more selective PDE inhibitors for diseases including sepsis, female sexual dysfunction, cardiovascular issues, and neurological disorders.
Cyclic AMP is a second messenger that mediates hormone signaling in cells. It is synthesized from ATP by adenylate cyclase in response to hormones like epinephrine and glucagon. Cyclic AMP activates protein kinase A, which causes effects like glycogenolysis, lipolysis, modulation of gene transcription, hormone secretion, increased gastric acid secretion, and regulation of cell permeability to water.
Protein kinase A (PKA) is an enzyme involved in the cAMP-dependent pathway. It is activated when cyclic AMP (cAMP) levels rise in response to stimuli like hormones or neurotransmitters. PKA then directly phosphorylates other proteins to regulate various cellular processes either rapidly through direct phosphorylation or slowly through activating transcription factors. PKA activity is regulated by anchoring proteins and feedback loops, and it functions in many tissues to modulate processes like metabolism, ion transport, gene expression, and more.
This document discusses various ways that enzyme activity can be regulated. It describes how enzyme levels and activity can be increased or decreased through different mechanisms, including substrate availability, product accumulation, allosteric effectors, covalent modification, genetic controls, zymogens, isozymes, and modulator proteins. A key example discussed is allosteric regulation, where binding of an allosteric effector at a site other than the active site can increase or decrease an enzyme's catalytic efficiency. Phosphorylation is provided as a common example of covalent regulation, where the addition of phosphate groups can alter an enzyme's activity.
The document summarizes phosphodiesterases (PDEs), which are important regulators of signal transduction mediated by the second messengers cAMP and cGMP. There are 11 PDE families with over 50 isoforms that degrade these cyclic nucleotides in different tissues. PDE inhibitors prevent degradation of cAMP and cGMP, classified as non-selective or selective. Selective PDE5 inhibitors like sildenafil are used to treat erectile dysfunction by potentiating the effect of nitric oxide in the penis.
Receptor tyrosine kinases (RTKs) are cell surface receptors that bind polypeptide growth factors, cytokines, and hormones. They regulate normal cellular processes but also play a critical role in cancer development and progression. There are approximately 20 classes of RTKs that exist as single or multimeric complexes and activate intracellular signaling pathways through autophosphorylation following ligand binding. Mutations in RTKs and their downstream effectors can lead to uncontrolled cell growth by constitutively activating growth signaling pathways. Several RTK inhibitors have been developed for cancer treatment, including those that target specific kinases as well as multi-kinase inhibitors.
This study investigated the functional redundancy of the four aspartic proteinases (plasmepsins) found in the digestive vacuole of the malaria parasite Plasmodium falciparum. The researchers disrupted each of the plasmepsin genes (PfPM1, PfPM2, PfPM4, and PfHAP) through genetic engineering. They found that while disruption of PfPM1 and PfPM4 resulted in reduced parasite growth, none of the plasmepsins were essential for survival. This suggests the plasmepsins can compensate for each other, likely due to their structural and catalytic similarities. The study implies that effective antimalarial drugs will need to inhibit multiple plasmepsin family members.
1. The authors performed a comparative phosphoproteomic analysis of host cells infected with wild-type Francisella novicida or a delta-lpcC mutant strain.
2. They found that actin and intermediate filaments/microtubules are important for F. novicida entry into host cells.
3. Infection with the delta-lpcC mutant induced hyper-phosphorylation and inhibition of the protein tristetraprolin, leading to increased production of cytokines like IL-1beta and TNF-alpha that can kill host cells.
Malaria vaccines cum antimalaria drugs, by bdollarbernard bahaah
This presentation slides give an upto date information on antimalaria drugs and vaccines. It can be used for academic purpose or some other purpose. It highlights some of the successes and potentials of antimalaria drugs.
This document discusses P-glycoprotein (P-gp), an ATP-dependent efflux pump found in the cell membranes of many tissues. P-gp pumps many foreign substances, drugs, and toxins out of cells. It plays an important physiological role and contributes to multidrug resistance in cancer cells by transporting chemotherapy drugs out of the cells. The document outlines the structure, mechanism of action, substrates, inhibitors, and approaches to bypassing P-gp efflux, such as using nanocarrier drug delivery systems.
This document summarizes a study that identified genes in the extracellular matrix that regulate the susceptibility of cultured cells to prion infection. The study compared gene expression in prion-susceptible and resistant cell lines. They identified 9 genes, including fibronectin 1 and integrin α8, that were upregulated in resistant cells. Knockdown of these genes increased susceptibility by altering the extracellular matrix structure and deposition of prion proteins. The results suggest the extracellular matrix plays a key role in controlling prion infection by influencing how prion proteins interact and convert forms.
1. The study found that the human prion protein (PrPc) has antimicrobial activity against both bacteria and fungi.
2. This antimicrobial activity is mediated by the unstructured N-terminal region of PrPc.
3. Synthetic peptides based on the PrPc N-terminus were shown to kill both Gram-positive and Gram-negative bacteria as well as the fungus Candida parapsilosis.
1. Elephants are considered for cancer research because they have an extremely low incidence of cancer despite their large body size and long lifespan, which typically increase cancer risk in other mammals.
2. Elephants may have evolved enhanced cancer resistance mechanisms due to evolutionary pressures from their large body size and long lifespans. Their cells appear to better repair DNA damage before mutations can cause cancer.
3. Elephants possess 20 copies of the p53 tumor suppressor gene, which is important for DNA repair and apoptosis, compared to single or double copies in most other mammals. Multiple copies of p53 may enhance elephants' ability to
This document discusses phospholipase C (PLC) and its role in neutrophil degranulation and T cell cytotoxicity. PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) into two second messengers, diacylglycerol and inositol 1,4,5-trisphosphate. PLC signaling is required for lytic granule polarization and effective T cell killing through T cell antigen receptor activation. Neutrophil degranulation involves increases in calcium and PLC production of phosphatidylinositol, which is essential for granule exocytosis. Different intracellular signaling pathways regulate the release of primary, secondary, and tertiary granules from neutroph
This document summarizes research investigating the binding affinity of four fungal peptides (Pneumocandin B0, Aureobasidin G, WF 11899A, and Echinocandin B) to the protein cFLIP using molecular docking software. The key findings were:
1) Pneumocandin B0 showed the highest binding affinity to cFLIP with a binding energy of -455.18.
2) Aureobasidin G had the second highest binding affinity with an energy of -414.96.
3) All four fungal peptides showed potential to inhibit cFLIP and could enhance TRAIL-mediated apoptosis in cancer cells, though further in vitro and in vivo studies
Environmental Factor - July 2014_ Intramural papers of the monthXunhai 郑训海
The document summarizes 5 research papers from the National Institute of Environmental Health Sciences (NIEHS). It describes the key findings and conclusions from each paper which include: 1) NIEHS developed a 7-step framework for systematic reviews to address environmental health questions. 2) A study found that polymerase beta can complement aprataxin function during DNA repair. 3) Retinoic acid-related orphan receptor gamma regulates hepatic glucose metabolism and insulin sensitivity. 4) The INO80 complex maintains embryonic stem cell pluripotency and regulates blastocyst development. 5) A study characterized structural changes in HIV reverse transcriptase formation providing insights for new therapeutics.
The role of natural products in regulating pyroptosisLucyPi1
This document reviews the role of natural products in regulating pyroptosis. It begins with background on pyroptosis, describing it as a form of inflammatory programmed cell death. It then discusses how pyroptosis is involved in various diseases and its molecular pathways. The document categorizes 14 natural products that have been shown to affect pyroptosis, including flavonoids like dihydromyricetin and terpenoids like andrographolide. These natural products were found to negatively or positively impact pyroptosis pathways by regulating factors like caspase-1, gasdermin D, and cytokines. The review concludes that natural products have potential as sources of new drugs for diseases related to uncontrolled pyroptosis.
Expression and Purification of the Plasmodium berghei Apical Membrane Antigen-1Nathan Jones
This document summarizes the production and purification of the Plasmodium berghei Apical Membrane Antigen-1 (PbAMA-1) protein for use as a vaccine candidate in mice. The PbAMA-1 gene was amplified from P. berghei genomic DNA and cloned into an expression vector. This plasmid was transformed into E. coli cells for protein expression. The expressed PbAMA-1 protein was purified using nickel affinity and size-exclusion chromatography. Analysis by SDS-PAGE and western blot confirmed the purified protein had the correct size and identity. The purified PbAMA-1 protein is now being tested in mice for its ability to protect against blood stage and sporozoite stage
Involvement of Interleukin-6 induced PI3K/Akt/mTor pathway in the regulation ...eshaasini
Hepatocellular Carcinoma (HCC) is an invasive cancer. Alphafoetoprotein (AFP) is a diagnostic marker for HCC directly related to the disease agressivity. Telomerase, is expressed by 90% of HCC. PI3K/Akt/mTOR pathway wich is regulated by IL-6 is activated in the HCC. Our aim is to investigate the effect of IL-6 on AFP and telomerase secretion in HepG2/C3A and PLC/ PRF/5 cell lines.
Involvement of Interleukin-6 Induced PI3K/Akt/mTor Pathway in the Regulation ...semualkaira
Hepatocellular Carcinoma (HCC) is an invasive
cancer. Alphafoetoprotein (AFP) is a diagnostic marker for HCC
directly related to the disease agressivity. Télomérase, is expressed
by 90% of HCC. PI3K/Akt/mTOR pathway wich is regulated by
IL-6 is activated in the HCC. Our aim is to investigate the effect
of IL-6 on AFP and telomerase secretion in HepG2/C3A and PLC/
PRF/5 cell lines.
Involvement of Interleukin-6 induced PI3K/Akt/mTor pathway in the regulation ...semualkaira
Hepatocellular Carcinoma (HCC) is an invasive cancer. Alphafoetoprotein (AFP) is a diagnostic marker for HCC directly related to the disease agressivity. Telomerase, is expressed by 90% of HCC. PI3K/Akt/mTOR pathway wich is regulated by IL-6 is activated in the HCC. Our aim is to investigate the effect of IL-6 on AFP and telomerase secretion in HepG2/C3A and PLC/ PRF/5 cell lines.
Involvement of Interleukin-6 induced PI3K/Akt/mTor pathway in the regulation ...eshaasini
Hepatocellular Carcinoma (HCC) is an invasive cancer. Alphafoetoprotein (AFP) is a diagnostic marker for HCC directly related to the disease agressivity. Telomerase, is expressed by 90% of HCC. PI3K/Akt/mTOR pathway wich is regulated by IL-6 is activated in the HCC. Our aim is to investigate the effect of IL-6 on AFP and telomerase secretion in HepG2/C3A and PLC/ PRF/5 cell lines.
Involvement of Interleukin-6 induced PI3K/Akt/mTor pathway in the regulation ...semualkaira
Hepatocellular Carcinoma (HCC) is an invasive cancer. Alphafoetoprotein (AFP) is a diagnostic marker for HCC directly related to the disease agressivity. Telomerase, is expressed by 90% of HCC. PI3K/Akt/mTOR pathway wich is regulated by IL-6 is activated in the HCC. Our aim is to investigate the effect of IL-6 on AFP and telomerase secretion in HepG2/C3A and PLC/ PRF/5 cell lines.
The document reports on the project to express and purify the methyltransferase domain (MTD) of TGS1 from Plasmodium falciparum (PfTGS1) in E. coli. The objectives are to clone the MTD of PfTGS1 into the pET43a expression vector, express the protein in E. coli BL21DE3 cells, and purify the protein using Ni-NTA beads. The strategy involves cloning the MTD, expressing the protein in E. coli, and purifying it using Ni-NTA beads. The materials and methods section describes finding the PfTGS1 gene and protein sequences, designing primers to amplify the MTD, cloning the M
Phosphatidylinositol 4-Kinase Enzymes: Functional Roles and Potential for Dru...IOSRJPBS
The two types of phosphatidylinositol 4-kinases (PI-4Ks) synthesize phosphatidylinositol 4-phosphate (PI-4P), a member of the phosphoinositide family. Phosphoinositides (PIPs) are synthesized from phosphatidylinositol (PI), a lipid containing the myo-inositol head group. PI can be phosphorylated at positions 3, 4, and 5 of the inositol ring which allows for seven different PIPs. Indeed, all of these enzymes have been identified in the cell. For instance, one prominent function of PIPs is to serve as membrane markers typically in concert with organelle specific proteins. PI(4,5)P2 is the main lipid determinant of the plasma membrane and PI3P and PI(3,5)P2 of the early and late endosomes. PI-4P is the main lipid determinant of the Golgi and trans-Golgi network (TGN) but, additionally, helps to define the specific character of the plasma membrane. This article reviews the recent developments in research on these enzymes and their potential for drug target.
Similar to Identification of Potent Phosphodiesterase Inhibitors that Demonstrate Cyclic Nucleotide-Dependent Functions in Apicomplexan Parasites (20)
2. Recently, it has been determined that three second
messenger signaling pathways control parasite egress and
reinvasion of host cells.3−6
An increase in cytoplasmic
concentration of the secondary messengers cyclic-AMP
(cAMP), cyclic-GMP (cGMP), and calcium ions (Ca2+
),
typically causes changes in the cellular program by activating
kinases through their direct or indirect binding. This results in
phosphorylation of specific substrates, which ultimately leads to
a change in cellular function. cAMP, for example, is a known
activator of Protein Kinase A (PKA), and this enzyme has been
implicated in the phosphorylation of the cytoplasmic tail of the
parasite adhesin Apical Membrane Antigen 1 (AMA1), which is
essential for P. falciparum erythrocyte invasion.5
Protein Kinase
G (PKG) on the other hand is directly activated upon a rise in
cGMP concentration, and in apicomplexan parasites this
enzyme appears to occupy an important nexus during multiple
transition stages within P. falciparum4,7−9
and during egress and
motility of Toxoplasma.6,10,11
Given that PKA and PKG require
a rise in intracellular concentration of cAMP and cGMP,
enzymes that control the production and degradation of these
cyclic nucleotides likely play a critical role in the activation of
egress and invasion. 3′-5′-Cyclic nucleotide phosphodiesterases
(PDEs) hydrolyze cAMP and cGMP into AMP and GMP,
respectively, thus attenuating their cellular accumulation.
Indeed, physiological PDE inhibition (eg, by regulatory domain
phosphorylation) has been described as a mechanism to
increase cAMP and cGMP levels, consequently activating
downstream events.12
Small molecule inhibitors of parasite
PDEs could therefore assist in the determination of events that
activate egress and invasion in apicomplexan parasites.
The human PDE5 (hPDE5) inhibitor, zaprinast (1; Figure
1), was the first compound identified as an inhibitor of parasite
PDEs.13
Specifically, it was shown to inhibit P. falciparum
proliferation (IC50 35 μM), inhibit recombinant P. falciparum
PDEα activity, and cause elevation of intracellular cGMP
levels.14
Zaprinast has also been used in the study of parasite
egress where it was shown to induce premature egress from
erythrocytes, which can be blocked by the PKG inhibitor,
Compound 1 (Cmpd1).4
Indeed, zaprinast can also induce
PKG-dependent egress in T. gondii.2
While zaprinast has been useful to determine the role of
PDEs in infection, and as a tool for activating parasite egress, it
is only a moderately potent inhibitor and therefore has limited
use. Several other PDE inhibitors were evaluated in the 2009
National Institute of Health (NIH) malaria screen,15
including
the nonisoform selective PDE inhibitor, Dipyridamole, and the
hPDE3 inhibitor Cilostazol with EC50 values against the 3D7
strain of approximately 10 μM. In addition, Beghyn and co-
workers originally explored the repurposing of the hPDE5
inhibitor, tadalafil (2; Figure 1) as a potential antiplasmodium
compound. The most potent tadalafil analogue (3; Figure 1)
had an EC50 of 0.5 μM.16
Recently, we reasoned that more potent parasite PDE
inhibitors might be identified by examining the homology at the
catalytic site of P. falciparum PDEs (PfPDE) as compared to
human PDEs,17
and using this we hypothesized that hPDE9
and hPDE1 isoforms were most like Pf PDE, thus providing a
mechanism to identify new apicomplexan PDE inhibitors. Here,
we have extended our examination of this topic now to the
apicomplexan parasite T. gondii PDEs (TgPDE). Furthermore,
we show that a series of compounds that inhibit hPDE1/9 elicit
much more potent cell responses than zaprinast on both P.
falciparum and T. gondii. We provide evidence as to the mode-
of-action of these new inhibitors, showing that they potently
inhibit recombinant Pf PDEα and induce host cell egress in
both P. falciparum and T. gondii, which can be blocked by
inhibiting PKG, and this is likely due to their ability to potently
activate microneme secretion. Interestingly, we also show that
our new PDE inhibitors act to induce an accumulation of
cAMP during AMA1 tail phosphorylation, thus suggesting that
the PDE target(s) of these compounds breaks down both
cAMP and cGMP during egress, motility, and invasion in
apicomplexan parasites. Together, these data describe potent
new compounds that can be used to study cyclic nucleotide
signaling across the Apicomplexa, which in time could offer new
insights for future disease control therapies.
■ RESULTS AND DISCUSSION
Homology of Pf PDEs and TgPDE. Previous work
demonstrating that zaprinast and other compounds are active
against P. falciparum and T. gondii suggested that these parasites
rely on one or more PDEs for critical cellular processes.2,4
The
P. falciparum genome encodes four highly similar PfPDEs, of
which only two, Pf PDEα and Pf PDEβ, appear to be expressed
in asexual blood stages. It is this stage that produces
symptomatic disease and is therefore the stage that most
PDE inhibition studies have targeted. Collins et al. suggested
Pf PDEβ as a good candidate for zaprinast inhibition,4
but the
Pf PDEs are highly homologous to one another at the active
site, and it is conceivable that it inhibits multiple isoforms. The
Pf PDEs are also homologous to the human targets of zaprinast
hPDE1, -5, and -9.17
Yuasa et al. showed that zaprinast inhibits
Pf PDEα with an IC50 of 3.8 μM, and in their studies of
Pf PDEδ knockout gametocytes, Taylor et al. showed that
Pf PDEδ is relatively insensitive to zaprinast whereas the
residual activity is zaprinast-sensitive.13,18
To extend this work, we wished to determine the PDE
target(s) of zaprinast in T. gondii. We retrieved 18 putative PDE
sequences from ToxoDB, the T. gondii genome database, and
reviewed the homology to human and Plasmodium sequences.
When aligned to the Pf- and hPDE sequences,
TgME49_202540 showed good homology to Pf PDEβ (27%)
and hPDE1B (25%) and in particular showed 72% homology at
the cyclic nucleotide binding site with Pf PDEβ (Table 1) and
matched hPDE1/9 nucleotide-binding residues as well at a
number of conserved amino acid positions. Other T. gondii
sequences also showed sufficient homology to the hPDE and
Pf PDE sequences to be considered candidate PDEs and
include TgME49_266920 [Pf PDEβ (26%) and hPDE1 (28%)]
and TgME49_228500 [Pf PDEβ (29%) and hPDE1 (33%)]
(data not shown).
In developing homology models of the PfPDE isoforms,17
we identified some key features that we believed would dictate
Figure 1. Structures zaprinast (1), tadalafil (2), and tadalafil analogue
(3).
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B
3. the potency of inhibitors of those PDEs, and these were found
to be conserved in the T. gondii orthologues (Table 1). In
particular, the residue adjacent to the conserved “purine-
scanning” glutamine is small in hPDE9 (A452
), hPDE1, Pf PDEβ
(S283
, Figure 2), and Tg49_202540, compared to a bulky M in
most human isoforms (Table 1 and Figure 2). We had also
identified a residue in the core pocket (N405
in hPDE9) as H or
N in the various parasite sequences (H235
in PfPDEβ, Figure 2)
as well as hPDE1, -4, -7, -8, and -9, but A in hPDE5. The larger
H residue in PfPDEs may explain why the hPDE5 inhibitor
sildenafil is a much less potent inhibitor in P. falciparum
proliferation assays relative to zaprinast. Finally we identified a
common hydrophobic pocket formed by three residues (L421
,
Y424
, and F441
of hPDE9 ; L251
, F254
, and L271
in Pf PDEβ, Figure
2). Overall, the similarity of key nucleotide binding residues of
hPDE1 or hPDE9 with the parasite PDEs supported inhibitors
of human hPDE1 and/or -9 as potential starting points from
which to develop parasite PDE inhibitors. We speculated that a
series of reported 1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one
hPDE1/9 inhibitors described by De Ninno et al. displayed
the requisite binding pharmacophore (Figure 2).19
Subse-
quently, an analogous 1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one
compound in cocrystal with hPDE9 was reported adopting the
pose as modeled by us.19,20
Discovery of 5-Benzyl-3-isopropyl-1H-pyrazolo[4,3-d]-
pyrimidin-7(6H)-one (6). Given that both P. falciparum and
T. gondii most closely resemble hPDE1/9 we prepared a
focused library of compounds based upon a series of already
reported inhibitors of this enzyme.19
The synthesis of all the
compounds derive from the key precursor 4 prepared by
adaptation of literature methods (Scheme 1). Condensation of
4 with various carboxylic acids using PyBroP under microwave
conditions yielded intermediate amides 5, which underwent
base catalyzed cyclization to yield the desired pyrazolopyr-
imidinones (6−23).21
An N-methyl derivative 24 was prepared
by direct methylation of 6 (see Supporting Information).
The compounds were first tested for their growth inhibitory
activity in cultured asexual blood stage P. falciparum parasites
Table 1. Alignment of Human, Pf, and Tg PDE Catalytic Site Residues Based upon Full Sequence Alignment
residuea
regionb
hPDE1 hPDE5 hPDE9a
PfPDEβa
TgME49_202540
292 M H H H292
H119
H
293 M D D D293
D120
D
296 M H H H296
H123
H
322 M E E E322
E149
E
325 M H H H325
H152
H
402 M D D D402
D232
D
251 Q Y Y F251
Y78
Y
405 Q H A N405
H235
N
413 Q H Q A413
H243
H
420 Q + H L V L420
V250
L
423 Q E E E423
E253
E
453 Q Q Q Q453
Q284
Q
456 Q F F F456
F287
F
490 Q W W Y490
W322
W
421 H M A L421
L251
S
424 H F F Y424
F254
F
441 H L L F441
L271
L
301 L N N N301
N128
N
302 L N S T302
L129
A
303 L F Y Y303
F130
L
452 L S M A452
S283
S
455 L G G G455
T286
G
459 L F A F459
F290
F
406 P I E406
G236
C
417 T A V417
C247
C
a
Numbering based upon hPDE9 X-ray structure PDB code 3DYN and Pf PDEβ model based on ref 17. b
Region guide M = metal binding, Q = core
pocket, H = hydrophobic pocket, L = Lid region (see ref 35). Shaded residue positions are discussed in the text.
Figure 2. Homology model of PfPDEβ catalytic site modeled with
BIPPO (6) by analogy to hPDE9 cocrystal (PDB: 3JSI). Residues are
highlighted which may dictate selective interactions between inhibitors
and binding site including Q284 conserved glutamine (Q453 in
hPDE9), H243 (A413), H235 (N405), S283 (A452), L251 (L421),
F254 (Y424), L271 (F441), and H277 (V447).
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4. by measuring the activity of the parasite enzyme lactate
dehydrogenase, after 72 h of parasite cultivation with the
inhibitors (Scheme 1 and Table 2). Compounds 6−11 showed
the most potent growth inhibitory activity in that order.
Compound 6, or 5-benzyl-3-isopropyl-1H-pyrazolo[4,3-d]-
pyrimidin-7(6H)-one, which we abbreviated to “BIPPO” and
with no substitution on the phenyl ring, was the most effective
compound with an IC50 of 0.4 μM in P. falciparum. The
potency of BIPPO was followed by compounds 7 and 8,
indicating that the respective para-chloro and para-fluoro
substitutions were tolerated (Scheme 1 and Table 2). The
presence of other substituents, shorter or longer links to the
aryl substituent, reduced potency as did, most notably,
methylation at the 7-positon (24) consistent with the proposed
pharmacophore.
Proliferation inhibitory studies were performed also with T.
gondii tachyzoites growing in human fibroblasts. Growth
potential was qualitatively measured by the number and size
of zones of host cell clearance (plaques) created by the lytic
growth of T. gondii. The three best compounds, as determined
by P. falciparum growth inhibition studies (above), had their
ability to block T. gondii growth determined. BIPPO showed
effective inhibition at concentrations as low as 2 μM and was at
least 30 times more effective than zaprinast at reducing plaque
formation (Figure 3). Just as for P. falciparum, compounds 7
and 8 were the next most potent at reducing tachyzoite
proliferation, and all were much more effective than zaprinast
(Figure 3). Note that the IC50 cannot be calculated using this
plaque assay as a readout.
BIPPO, 6, with its robust activity in both growth inhibition
assays appeared to be the compound to investigate further to
establish parasite PDE inhibition as the mechanism of action.
We first attempted to confirm that BIPPO was a potent
inhibitor of Pf PDEs by testing it against recombinant enzymes.
We expressed PfPDEα with a glutathione S-transferase (GST)
tag in Escherichia coli and obtained a partially purified protein
(Supporting Information Figure s1) that showed robust
hydrolysis of cGMP. While full characterization of the enzyme
and inhibitors was not possible due to its poor stability, it was
found that BIPPO inhibited this activity in a dose dependent
manner, with an IC50 of approximately 150 nM, while zaprinast
was much less potent at 5 μM affording only approximately
40% inhibition, (consistent with the report of Yuasa et al.;13
Supporting Information Figure s2). We also observed that the
enzyme showed modest hydrolytic activity against cAMP
(Supporting Information Figure s3), and when both cGMP
and cAMP were in the same reaction, hydrolysis of cGMP was
virtually complete while significant cAMP remained (Support-
ing Information Figure s4). We also attempted to prepare
recombinant Pf PDEβ, and while observing some activity
against cGMP the enzyme was even less stable than PfPDEα,
making reproducible measurements problematic, and the data
Scheme 1. Synthesis of Compounds 6−24 (Top) and
Structures of R Groups (Bottom)
Table 2. Antiproliferative Activity of the
Pyrazolopyrimidinones against the Asexual Blood Stage of
Plasmodium falciparum 3D7 Strain
compound IC50 (μM)
1, zaprinast 35 ± 4.2a
6 (BIPPO) 0.40 ± 0.14b
7 0.64 ± 0.21
8 0.73 ± 0.30
9 0.74 ± 0.23
10 1.2 ± 0.24
11 2.4 ± 0.6
12 2.9 ± 0.6
13 3.6 ± 0.85
14 4.7 ± 1.9
15 7.8c
16 5.6
17 10
18 11.5 ± 3.7
19 25
20 35 ± 6.7
21 40
22 >100
23 >100
24 70
a
Reference 13. b
IC50 ± SEM (n = 3 or 4). c
IC50 (n = 2).
Figure 3. PDE inhibitors preventing T. gondii growth. Parasites
incubated with confluent HFF cells for 7 days with indicated
concentrations (μM) of PDE inhibitors. Each zone of clearance
(white plaques) represents one tachyzoite undergoing repeated rounds
of invasion, replication, and egress. T. gondii is more sensitive to new
PDE inhibitors than to zaprinast with plaques prevented at
concentrations upward of 55 μM of PDE inhibitors tested. Scale bar
= 5 mm.
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D
5. will not be presented here. BIPPO was consequently also
screened against recombinant hPDE isoforms and confirmed
potent hPDE9 activity (IC50 = 30 nM), with significant
inhibition at 1 μM against hPDE1 (42%), hPDE5 (66%), and
hPDE6 (64%). Attempts to directly measure the increase in
levels of these cyclic nucleotides in cultured blood stage P.
falciparum, using commercial microplate kits, were unsuccessful
since nucleotide levels were too low to reliably measure (data
not shown).
BIPPO Potently Activates Parasite Egress in P.
falciparum and T. gondii. We assessed the activity of
BIPPO in comparison to zaprinast in a number of cyclic
nucleotide dependent functional assays in both P. falciparum
and T. gondii. To quantitatively measure egress in P. falciparum
cultures, we created an egress reporter parasite line by
transfecting the 3D7 strain with a highly active luciferase called
NanoLuc fused to a signal peptide sequence at its N-
terminus.22,23
The signal peptide promotes secretion of
NanoLuc into the parasitophorous vacuole (PV) space that
surrounds the intracellular parasite. Just prior to egress, the
parasite breaks down the PV membrane and the plasma
membrane of the erythrocyte host, and the extent to which this
occurs can be ascertained by measuring the levels of luciferase
released into the supernatant. After 20 min of incubation,
BIPPO induced greater egress at substantially lower concen-
trations than zaprinast and induced a 2.5 fold higher egress
peak at saturating levels of the compounds (Figure 4a).
To determine if the egress response seen in P. falciparum due
to PDE inhibition was also seen in T. gondii, we monitored
egress response to a titration of our panel of compounds and
compared their effectiveness to zaprinast. Egress was measured
as a function of host cell lactate dehydrogenase (LDH) release,
which occurs due to damage created by egressing tachyzoites.2
In comparison to zaprinast, T. gondii egress was more sensitive
to five compounds (BIPPO, 7, 8, 10, and 12, Scheme 1 and
Table 2); one compound (18) showed a similar response to
zaprinast (1), and one compound was ineffective at inducing
tachyzoite egress (15; Figure 4b−h). The data largely correlates
with the activity shown by the compounds in the T. gondii
proliferation (plaque) assay (Figure 3).
To understand further the effect that PDE inhibition has on
parasite egress, we undertook live cell imaging of both P.
falciparum and T. gondii. Zaprinast has been recently shown to
induce premature egress in asexual, late stage blood cultures of
P. falciparum with an EC50 of 25 μM4
and to also activate
tachyzoite egress from host cells in T. gondii.2
To confirm that
BIPPO was actually inducing the breakdown of the PV
membrane and the plasma membrane of infected erythrocytes,
time-lapse imaging was performed on late stage parasites. Over
a 30 min period, 2 μM BIPPO appeared to induce the
breakdown of the erythrocyte and vacuole membranes around
fully formed merozoites, but they could not invade the
surrounding erythrocytes presumably because either they
were not developmentally mature or because BIPPO also
inhibited the invasion process (Figure 5A, left; Movie 1).
Immature parasite forms undergoing cytokinesis were also
released, highlighting the fact that BIPPO was accelerating
breakdown of the host before the merozoites were mature,
presumably through an increase in cGMP levels (Figure 5a,
right; Movie 1). Zaprinast induced egress was not undertaken
since it has been performed previously.4
To investigate the response of T. gondii to these new
inhibitors, BIPPO and zaprinast were compared for egress
induction via live microscopy (Figure 5B and Movie 2).
Tachyzoites were allowed to invade and replicate for 30 h to
produce vacuoles with >16 parasites. Tachyzoite egress was
seen rapidly after the addition of the compounds (Cf =55 μM)
Figure 4. BIPPO (compound 6) inducing parasite egress. (a) In P.
falciparum blood stages, BIPPO more efficiently induces egress than
zaprinast through being more active at lower concentration and
achieving higher peak activity after 20 min of incubation. Egress was
quantified by measuring luciferase activity as relative light units (RLU)
in the growth media of parasites induced to breakdown their
enveloping vacuole and erythrocyte membranes, thereby releasing
the luciferase. The data points represent mean ± SD, n = 3. (b−f)
Tachyzoite egress following 5 min treatment with different
concentrations of PDE inhibitors was measured as a function of
lactate dehydrogenase release from host cells, and normalized to
maximal egress. Each plot displays indicated compound treatment
compared with zaprinast. Mean ± S.E.M, n = 3. Plots indicate that
compounds BIPPO (b), C7 (c), C8 (d), C12 (e), and C10 (f)
outperform zaprinast as an egress stimulant, while C18 (g) is
comparable to zaprinast and C15 (h) is ineffective.
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E
6. with BIPPO stimulating egress at 1:30 min faster than zaprinast
(5:30). Compounds 7 and 8 were also tested, and they too
induced egress with rapidity consistent with the compound
curves (Supporting Information Figure s5). It is also worth
noting that T. gondii and P. falciparum undergo intracellular
replication in a different manner. T. gondii undergoes binary
division and is always ready to egress; thus premature egress is
not possible.
PKG Inhibitor Can Block BIPPO Indicating Specificity
for a cGMP-Dependent PDE. In other systems, cGMP can
function by activating ion channels and promoting phosphor-
ylation via PKG. If BIPPO and the other PDE inhibitors exert
their egress-promoting effects in a PKG-dependent processes,
then inhibition of this kinase should block the egress-promoting
effects of BIPPO. Compound 1 (Cmpd1) is a specific inhibitor
of apicomplexan PKG,11
and it has been shown that Cmpd1
blocks the effects of zaprinast in both P. falciparum and T.
gondii.2,4,24
To determine if Cmpd1 could therefore block the
pro-egress effects of BIPPO, the P. falciparum NanoLuc
reporter parasites were incubated ±2.5 μM of Cmpd1 along
with the approximate IC50 levels of BIPPO (0.7 μM) and
zaprinast (40 μM) for 0, 10, 20, and 40 min (Figure 6A). Under
these conditions, Cmpd1 efficiently blocked the egress-
promoting effects of zaprinast and BIPPO, confirming the
latter is also a cGMP PDE inhibitor (Figure 6A).
We performed parallel experiments in T. gondii, which is
susceptible to zaprinast in a Cmpd1-dependent manner.2
After
pretreatment of intracellular tachyzoites with a titration of
Cmpd1, parasites were stimulated to egress using a fixed
concentration of our compounds (BIPPO, 7, 8, and zaprinast)
(Figure 6b−d), while increasing the concentration of Cmpd1.
The most potent PDE inhibitor, BIPPO, required greatest
concentrations of Cmpd1 to inhibit egress than 7, 8, and
zaprinast. Furthermore, as visualized by live video microscopy,
Cmpd1 pretreatment completely ablated the tachyzoite
response to the C7, C8 and BIPPO compounds, showing a
Figure 5. Movie stills showing rapid egress of Plasmodium falciparum and Toxoplasma gondii induced by BIPPO (added at black arrow). (a, left)
Sequential video images showing P. falciparum merozoite egress (red box) beginning 10:30 after the addition of 2 μM BIPPO. (Right) After 15:30,
BIPPO induces breakdown of membranes surrounding the doubly infected erythrocyte before the schizonts have undergone complete cytokinesis.
Time stamp is minutes:seconds. Scale bar = 5 μm. (b) Intracellular T. gondii tachyzoite vacuoles (outlined by dashed white line) were exposed to
either zaprinast (500 μM) or BIPPO (55 μM) at 30 s (arrow). BIPPO-treated T. gondii respond much more rapidly and at a lower concentration
than zaprinast treatment. Scale bar = 20 μm.
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F
7. complete lack of response even after 10 min of stimulation
(Supporting Information Figure s5, Movie 3).
PDE Inhibitors Stimulate the Release of Secretory
Organelles in Toxoplasma. Host cell egress and motility in
apicomplexan parasites relies on the secretion of adhesins and
perforin-like molecules from the microneme organelles, which
is controlled by intracellular calcium and PKG signaling
events.6,11,25
Given that our new PDE inhibitors rapidly
stimulate host cell egress in both T. gondii and P. falciparum,
we hypothesized that this was due to the activation of
microneme secretion. To test this, we applied the inhibitors
to a well-established semiquantitative microneme secretion
assay in T. gondii6,25
(this assay is not well developed for P.
falciparum and hence could not be used here). Here, the
amount of proteolytically cleaved Mic2 (a major Toxoplasma
adhesin) is measured in the supernatant of extracellular
tachyzoites after stimulation and is used as a surrogate for
total microneme release. To do this, we treated extracellular
tachyzoites for 20 min with 500 μM of zaprinast or 55 μM of
BIPPO, 7, and 8. The supernatant (containing any excreted and
cleaved MIC2 in response to stimulation) and pellet fraction
were collected by centrifugation and then fractionated by SDS-
PAGE. Western blot was then used to determine the amount of
MIC2 present in the supernatant. The three PDE inhibitors
induced the secretion of MIC2 into the supernatant and appear
more potent over the given time frame than zaprinast (Figure
7). Overall, this provides further evidence that the new
compounds are much more potent PDE inhibitors than
zaprinast and act, at least in part, to induce parasite egress
through the stimulation of microneme secretion.
BIPPO Enhances PKA/cAMP-Dependent AMA1 Tail
Phosphorylation. Given the restricted expression of PDE
isoforms through the blood stage of the parasite in P.
falciparum, we hypothesized that the target(s) of BIPPO may
also break down cAMP. We have shown previously in P.
falciparum (not yet studied in T. gondii) that the cytoplasmic
tail of the merozoite invasion ligand apical membrane antigen 1
(AMA1) is phosphorylated by the cAMP-dependent protein
kinase A, at serine 610.5
To measure phosphorylation here, an
in vitro reaction was performed on the recombinant GST-
AMA1 tail in the presence of parasite lysate, [γ‑32
P] ATP, and 2
μM cAMP. To determine if BIPPO could block cAMP
hydrolysis, we added 6 μM to a series of in vitro
phosphorylation reactions containing 2, 1, 0.5, 0.25, and 0
μM cAMP (Figure 8 and Supporting Information). Without
BIPPO, the levels of AMA1 tail phosphorylation declined very
rapidly with decreasing cAMP concentrations, suggesting the
nucleotide was being rapidly hydrolyzed. The rate of
phosphorylation decline, however, could be substantially
inhibited by BIPPO, suggesting it acts against a cAMP-
degrading Pf PDE (Figure 8).
Given their greater potency and rapid action, BIPPO and its
analogues have great potential as tools to understand signaling
pathways mediating egress and invasion in Apicomplexan
parasites. Previously, use of calcium ionophores (ionomycin or
A23187) was standard for assessing active or defective
biological responses such as egress, motility, microneme
secretion, and invasion.3,6,25−27
However, this approach is
problematic as calcium ionophores induce a crude, global Ca2+
change in the intracellular environment by indiscriminately
releasing calcium from intracellular stores and allowing Ca2+
to
enter from external sources.28
This completely ablates the
intricacies and importance of calcium dynamics, such as calcium
flux, amplitude, and periodicity. Furthermore, signal pathway
induction by ionophores prohibits analysis of any events
Figure 6. cGMP-specific protein kinase G (PKG) inhibitor compound
1 (Cmpd1) blocking egress-promoting effects of BIPPO and other
putative PDE inhibitors confirming the inhibitors are targeting parasite
cGMP-specific PDEs. (a) Cmpd1 at 2.5 μM was able to block the pro-
egress effects of 0.7 μM BIPPO and 40 μM zaprinast upon NanoLuc
expressing P. falciparum blood stage parasites. Egress was measured via
luciferase present in the supernatant after host cell lysis. RLU was
plotted as a function of time for the various treatments (mean ± SD, n
= 3). (b−d) Inhibition of tachyzoite egress following 20 min
preincubation with different concentrations of Cmpd1 (Ctop 18 μM)
and 5 min treatment with a fixed concentration of PDE inhibitors (Cf
55 μM). Egress was measured as a function of lactate dehydrogenase
release from host cells and normalized to maximal egress. Each plot
displays indicated drug treatment compared with zaprinast. Mean ±
S.E.M., n = 3. Plots demonstrate egress response was inhibited by
Cmpd1 at a comparable rate to zaprinast.
Figure 7. PDE inhibitors stimulate microneme secretion. Extracellular
tachyzoites treated with zaprinast (Cf 500 μM) or PDE inhibitors (Cf
55 μM). Microneme secretion was determined by Western Blot
detection of secreted and cleaved micronemal protein MIC2 (cMic2)
relative to nonsecreted, full length MIC2 (fMIC2). Supn (super-
natant).
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8. preceding calcium release. Indeed, cGMP signaling has been
suggested to act before intracellular Ca2+
release and therefore
argues that the use of BIPPO provides a superior alternative to
current stimuli in a more biologically relevant environment.9
The combination of the use of BIPPO, Cmpd1, and Ca2+
ionophore may indeed help tease out the order of events that is
required for Apicomplexan host cell egress and help understand
how a change in extracellular K+
activates egress or how the
engagement of host cell receptors promotes invasion.
The consequences of inducing rapid and premature parasite
egress in an infected host are not absolutely clear, and so the
therapeutic potential of PDE inhibition remains to be
examined. On the other hand, dysregulation of key events in
host cell invasion such as AMA1 phosphorylation, that rely
upon cAMP dependent Pf PKA phosphorylation for efficiency,
may provide one mechanistic pathway to a therapeutically
useful application of PDE inhibition or render parasites more
susceptible to other drugs.
In conclusion, the broad homology between the mammalian
and parasitic PDE active site residues14
suggested that it should
be possible to repurpose hPDE inhibitors for use in studying
parasite signaling. Our approach to using homology modeling
to select the most homologous of the human enzymes for
parasite orthologues led us to develop BIPPO, which is much
more potent than the previously utilized tool compound
zaprinast, at inhibiting growth and inducing premature parasite
egress in a PKG-dependent manner.
While BIPPO shows potent inhibition of Pf PDEα consistent
with its observed cell-based activity, the specific Pf PDE and
TgPDE targets of BIPPO in parasites are still unknown, and the
effect on both cGMP and cAMP pathways suggests they may
emerge as being a single dual specificity PDE, multiple PDEs,
or a cGMP or cAMP-PDE involved in PDE cross-talk, and this
is a key goal of future work. The greater activity of PfPDEα
against cGMP over cAMP reflects the current order in which
we think these nucleotides function; i.e., cGMP stimulates
egress followed by cAMP dependent phosphorylation of
AMA1, which enables invasion. Optimizing the pharmaco-
logical inhibitors of parasite PDE functions may provide novel
compounds for the study of signal transduction processes
governing apicomplexan host cell egress and invasion and
identify new intervention points for therapy in these diseases.
■ METHODS
Plasmodium falciparum Strains and Transfections. P.
falciparum (strain 3D7) asexual blood stage parasites were
cultured as per ref 29 in RPMI-HEPES media supplemented
with L-glutamine (Sigma) and Albumax II (Invitrogen). To
express a luciferase enzyme that could be secreted into the
parasitophorous vacuole of infected red blood cells, DNA
sequence corresponding to 23 amino acid endoplasmic
reticulum signal sequence of merozoite surface protein 1 was
appended onto the 5′ end of the Promega NanoLuc sequence.
3D7 parasites were transfected with the NanoLuc construct by
culturing in erythrocytes that had been electroporated with 100
μg of the DNA30,31
and selected on 5 μg/mL blasticidin S.
Toxoplasma Culture. Toxoplasma was grown in Human
Foreskin Fibroblasts (HFFs) and passaged as required. Here,
HFFs were grown to confluency in DME supplemented with
10% Cosmic Calf Serum (Hyclone) and just before inoculation
with T. gondii media was refreshed and serum levels dropped to
1% fetal calf serum. All culture and assay conditions performed
at 37 °C, 10% CO2.
Plasmodium Growth Assay. Asexual blood stage parasites
12−24 h post infection (hpi) were grown for 72 h in
compounds serially diluted in DMSO (0.2% v/v in culture
media). Parasite growth was assessed by measuring the activity
of parasite lactate dehydrogenase using the Malstat assay.32
Toxoplasma Plaque Assay. Growth was determined by
the ability of tachyzoites to form zones of clearance (plaques)
in host cells through repeated cycles of invasion, replication,
and egress. This was done by adding tachyzoites to confluent
HFFs (Cf 100cells/ml) and treated with indicated concen-
trations of the test compound for the entirety of the assay.
Plates were left undisturbed for 7 days at 37 °C, fixed with 70%
methanol, stained with Crystal Violet, and imaged.
Plasmodium Egress Assays. 3D7_NanoLuc parasites23
were cultured until 44 h postinvasion at 5% parasitemia. The
culture was washed in RPMI medium to remove any NanoLuc
from the supernatant and resuspended at 2% hematocrit in
RPMI medium. Cmpd1 was added to half of the culture at 2.5
μM and 0.2 μL of zaprinast or BIPPO dilutions in DMSO were
added to 100 μL of Cmpd1-treated and -untreated culture in
duplicate. Samples were allowed to warm to 37 °C for 5 min
and then placed on ice after a 0, 10, 20, or 40 min incubation
period. Cells were pelleted at 3000g for 5 min; then 10 μL of
culture supernatant was mixed with 10 μL of NanoLuc assay
buffer (2× Promega cell lysis buffer and 1 μL Nano Glo
substrate per mL) in a luminometer plate, and the activity was
measured with a FluoStar Optima instrument (BMG Labtech).
Figure 8. BIPPO blocks the decline of cAMP-dependent phosphor-
ylation by inhibiting degradation of cAMP by PDEs in P. falciparum
extracts. (a) Diagram of recombinant GST-AMA1S610only tail fusion
protein that has had all its known phosphorylation sites mutated to
alanine (red) except for the S610 PKA site (blue). (b) An
autoradiograph of [γ32
P] labeled GST-AMA1S610only tail proteins that
have been phosphorylated by Pf PKA in lysates from P. falciparum
blood stage parasites. At lower levels of cAMP, phosphorylation
declines rapidly, presumably due to degradation by native PDEs, which
could be reversed by BIPPO. (c) Densitometry plots as a function of
cAMP concentration (mean ± SD, n = 3).
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9. Relative light units (RLU) were plotted as a function of
inhibitor concentration or time in Prism (GraphPad), using
nonlinear regression analysis as a sigmoidal dose−response
curve with variable slope. Samples were assayed in triplicate.
Live cell imaging of P. falciparum parasites was performed as
per ref 33 after the addition of 2 μM BIPPO.
Toxoplasma LDH Egress Assay. Egress was determined as
a function of lactate dehydrogenase (LDH) released from the
host cell as parasites egress.2
Tachyzoites (Cf 5 × 105
cells/ml)
were added to confluent HFFs in a 96 well plate format and
grown for 32 h. Wells were washed once with Ringers/5%FCS
before 1:3 titration of PDE inhibitors and zaprinast for 5 min at
37 °C (Ctop 500 μM). For Cmpd1 inhibition, cells were
pretreated with a titration of Cmpd1 (Ctop 18 μM) for 20 min
at 37 °C; then stimulated with PDE inhibitors (55 μM) or
zaprinast (500 μM) for 5 min at 37 °C. Supernatants were
taken and LDH detected using Promega CytoTox LDH assay
kit according to manufacturer’s instructions. Data were
normalized to 100% egress response in each assay. IC50 and
EC50 were determined using Prism (GraphPad) plotting
normalized, log transformed (x axis), nonlinear regression
analysis as a sigmoidal dose−response curve with variable slope.
Samples were assayed in triplicate for each assay, n = 3.
Treatments did not induced significant LDH release in the
absence of parasites (data not shown).
Toxoplasma Live Egress. Tachyzoites were added to
confluent HFFs in imaging chambers at 1 × 104
cells/mL and
grown for 30 h. In relevant instances, cells were pretreated with
2 μM Cmpd1 for 20 min at 37 °C. Wells were rinsed, and
media was replaced with Ringers/5%FCS. Cells were imaged
using a heated chamber at 37 °C with PDE inhibitors (55 μM)
and zaprinast (500 μM) added at 00:30 time-points. Images
were recorded for 10 min.
Toxoplasma Microneme Secretion Assay. Fresh extrac-
ellular cells (2 × 108
cells/mL) were treated for 20 min at 37
°C with PDE inhibitors (Cf 55 μM) and zaprinast (Cf 500 μM)
in 3%FCS DME. Microneme secretion was detected using
Western blot, probing for micronemal protein MIC2.34
■ ASSOCIATED CONTENT
*S Supporting Information
Movie 1: Treatment of Plasmodium falciparum blood stage
parasites with 2 μM BIPPO results in premature egress. Movie
2: Treatment of Toxoplasma gondii infected human fibroblasts
with 55 μM BIPPO triggers rapid parasite egress from their
host cells. Movie 3: Pretreatment of Toxoplasma gondii infected
human fibroblasts with 2 μM Cmpd1 blocks the egress
normally triggered by BIPPO. This material is available free of
charge via the Internet at http://pubs.acs.org.
■ AUTHOR INFORMATION
Corresponding Authors
*E-mail: Philip.Thompson@monash.edu.
*E-mail: tonkin@wehi.edu.au.
*E-mail: gilson@burnet.edu.au.
Author Contributions
#
These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
B.L.H., K.L.H., and R.J.S. are recipients of Australian
Postgraduate Awards, and C.J.T is recipient of an Australian
Future Fellowship (FT1200100164). This work was supported
by NHMRC Project grants 1025598 and 603720 as well as the
Victorian State Government Operational Infrastructure Support
Program and NHMRC IRIISS. We are grateful to the
Australian Red Cross for the supply of red blood cells. We
also thank K. Rogers for help with microscopy and T. Luc for
technical assistance.
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ACS Chemical Biology Articles
DOI: 10.1021/cb501004q
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