This document discusses kinase inhibitors and p38 MAP kinase as a drug target. It provides background on kinases and their role in disease. Many kinase inhibitors have been developed and some have been approved to treat cancers. The document discusses challenges with developing kinase inhibitors and strategies to increase selectivity, such as targeting inactive kinase conformations. It describes a compound called KC706 that was developed by Kemia to selectively inhibit p38α kinase in a time-dependent manner. KC706 shows potent and selective inhibition of p38α kinase activity and cytokine production in vitro.
This document provides information about Nanosyn, a contract research organization that offers various drug discovery services including high throughput screening, assay development, and medicinal chemistry services. It describes Nanosyn's microfluidic-based detection technology which allows simultaneous detection of enzyme substrates and products in a single sample run. This provides accurate and reproducible results. The technology also tolerates autofluorescent compounds and enables identification of both inhibitors and activators.
Delivering More Efficient Therapeutic Protein Expression Systems Through Cell...Merck Life Sciences
Historically cell line performance has been enhanced through media, feed and process optimization, primarily through trying to meet the basic nutritional requirements of the cells so that they can sustain high growth and productivity throughout the production runs.
However, the omics (genomics, transciptomics and metabolomics) era, sequencing of the CHO genome and enhancements in genome editing technologies over the past several years have enabled scientists to take a more direct route in cell line optimization through the modification of specific genes that have direct implications on cell culture performance, protein quality attributes and upstream and downstream manufacturing processes. These targets include but are not limited to genes that may be involved in cell cycle regulation, cellular metabolism, cellular transcription and translation, the secretory pathway and protein glycosylation or other post-translational modifications.
In this webinar we will discuss specific genetic modifications that have been made to CHO cell lines and how these modifications can lead to more efficient expression systems.
Vitamin A and its derivatives (retinoids) play an important role in cell growth, differentiation and apoptosis. They are obtained through diet as retinol, retinyl esters or beta-carotene and stored in the liver. Retinol is converted through a series of oxidation reactions to produce retinoic acid, which functions as a ligand for nuclear retinoid receptors and regulates gene expression. Retinoic acid also has non-genomic functions and can regulate pathways such as PI3K independently of nuclear receptors. Rheumatoid arthritis is associated with environmental and biological factors such as smoking, elevated tumor necrosis factor-alpha levels and abnormal B cell activity. TNF-alpha promotes inflammation and is a
Epidermal growth factor and its receptor tyrosine kinaseGedion Yilma
The document discusses epidermal growth factor (EGF) signaling and the EGF receptor. It notes that EGF is involved in normal cell processes like development, differentiation, and wound healing. The EGF receptor belongs to the ErbB family of receptor tyrosine kinases and plays a key role in signaling pathways regulating cell proliferation, survival, and apoptosis. Overexpression or abnormal activation of the EGF receptor and other ErbB family members is implicated in many epithelial cancers.
Cloning and Expression of recombinant ProteinGaurav Dwivedi
Protein G was cloned from streptococcal strains and expressed in E. coli BL21 cells. Various expression and purification conditions were optimized, including inducer type, temperature, media, and heat treatment. Protein G was purified using immobilized metal affinity chromatography and its ability to bind IgG subclasses was confirmed using a VersaFLo system. Heat treatment at 80°C removed contaminants and was an efficient step in downstream processing. Overall, the study successfully cloned and expressed recombinant protein G and analyzed its IgG binding properties.
Dna Methylation Analysis in a Single Day - Download the SlidesQIAGEN
This webinar introduces the new PyroMark Q48 Autoprep system. Combined with the latest EpiTect Fast bisulfite conversion technology, the new PyroMark Q48 Autoprep can now provide highly automated methylation analysis in a single day.
- PlcRa is a quorum sensing transcriptional regulator in Bacillus cereus that is activated by the heptapeptide PapRa7 during the transition to stationary phase.
- PlcRa upregulates genes involved in cysteine synthesis, oxidative stress resistance, and peptide transport while downregulating phage genes and the Hbl enterotoxin.
- PlcRa helps B. cereus adapt to poor sulfur conditions and oxidative stress early in stationary phase by controlling the regulator AbrB2 which in turn regulates genes for cysteine metabolism from methionine.
This document provides information about Nanosyn, a contract research organization that offers various drug discovery services including high throughput screening, assay development, and medicinal chemistry services. It describes Nanosyn's microfluidic-based detection technology which allows simultaneous detection of enzyme substrates and products in a single sample run. This provides accurate and reproducible results. The technology also tolerates autofluorescent compounds and enables identification of both inhibitors and activators.
Delivering More Efficient Therapeutic Protein Expression Systems Through Cell...Merck Life Sciences
Historically cell line performance has been enhanced through media, feed and process optimization, primarily through trying to meet the basic nutritional requirements of the cells so that they can sustain high growth and productivity throughout the production runs.
However, the omics (genomics, transciptomics and metabolomics) era, sequencing of the CHO genome and enhancements in genome editing technologies over the past several years have enabled scientists to take a more direct route in cell line optimization through the modification of specific genes that have direct implications on cell culture performance, protein quality attributes and upstream and downstream manufacturing processes. These targets include but are not limited to genes that may be involved in cell cycle regulation, cellular metabolism, cellular transcription and translation, the secretory pathway and protein glycosylation or other post-translational modifications.
In this webinar we will discuss specific genetic modifications that have been made to CHO cell lines and how these modifications can lead to more efficient expression systems.
Vitamin A and its derivatives (retinoids) play an important role in cell growth, differentiation and apoptosis. They are obtained through diet as retinol, retinyl esters or beta-carotene and stored in the liver. Retinol is converted through a series of oxidation reactions to produce retinoic acid, which functions as a ligand for nuclear retinoid receptors and regulates gene expression. Retinoic acid also has non-genomic functions and can regulate pathways such as PI3K independently of nuclear receptors. Rheumatoid arthritis is associated with environmental and biological factors such as smoking, elevated tumor necrosis factor-alpha levels and abnormal B cell activity. TNF-alpha promotes inflammation and is a
Epidermal growth factor and its receptor tyrosine kinaseGedion Yilma
The document discusses epidermal growth factor (EGF) signaling and the EGF receptor. It notes that EGF is involved in normal cell processes like development, differentiation, and wound healing. The EGF receptor belongs to the ErbB family of receptor tyrosine kinases and plays a key role in signaling pathways regulating cell proliferation, survival, and apoptosis. Overexpression or abnormal activation of the EGF receptor and other ErbB family members is implicated in many epithelial cancers.
Cloning and Expression of recombinant ProteinGaurav Dwivedi
Protein G was cloned from streptococcal strains and expressed in E. coli BL21 cells. Various expression and purification conditions were optimized, including inducer type, temperature, media, and heat treatment. Protein G was purified using immobilized metal affinity chromatography and its ability to bind IgG subclasses was confirmed using a VersaFLo system. Heat treatment at 80°C removed contaminants and was an efficient step in downstream processing. Overall, the study successfully cloned and expressed recombinant protein G and analyzed its IgG binding properties.
Dna Methylation Analysis in a Single Day - Download the SlidesQIAGEN
This webinar introduces the new PyroMark Q48 Autoprep system. Combined with the latest EpiTect Fast bisulfite conversion technology, the new PyroMark Q48 Autoprep can now provide highly automated methylation analysis in a single day.
- PlcRa is a quorum sensing transcriptional regulator in Bacillus cereus that is activated by the heptapeptide PapRa7 during the transition to stationary phase.
- PlcRa upregulates genes involved in cysteine synthesis, oxidative stress resistance, and peptide transport while downregulating phage genes and the Hbl enterotoxin.
- PlcRa helps B. cereus adapt to poor sulfur conditions and oxidative stress early in stationary phase by controlling the regulator AbrB2 which in turn regulates genes for cysteine metabolism from methionine.
Dasatinib is a second-generation BCR-ABL tyrosine kinase inhibitor that is more potent than imatinib and effective against some imatinib-resistant mutations. Unlike imatinib, dasatinib interferes with platelet function by targeting Src family kinases, which may contribute to increased bleeding risk in patients. Clinical trials found dasatinib achieved high rates of cytogenetic and molecular response in chronic myeloid leukemia patients, including those resistant to or intolerant of imatinib. However, dasatinib treatment also carried risks of myelosuppression and bleeding that required dose reductions in some cases.
Evaluation of the changes in the gene CYP3A4 expression in HepG2 cells under ...Angela Farngren
This study evaluated the effects of rifampicin and alpha-ketoglutarate on expression of the CYP3A4 gene in HepG2 liver cells. HepG2 cells were treated with either 10mM rifampicin or 4mM alpha-ketoglutarate for 7 days. RNA was extracted on days 0, 3, and 7 and analyzed via qPCR. Rifampicin treatment significantly increased CYP3A4 expression, peaking on day 3 and decreasing slightly by day 7. Alpha-ketoglutarate initially decreased CYP3A4 expression for the first 3 days but then increased expression levels. The study demonstrates that rifampicin and alpha-ket
Gamma-irradiated Frozen Cells Validated for GPCR-mediated Kinase SignalingPerkinElmer, Inc.
This document summarizes research from PerkinElmer and TGR Biosciences on using gamma-irradiated frozen cells for G protein-coupled receptor (GPCR) signaling assays. Specifically, it shows that frozen "cAMPZen" and "AequoZen" cells can be used with AlphaScreen SureFire assays to measure kinase pathways like ERK, Akt, and CREB activation downstream of various GPCRs. The assays performed similarly to cultured cells and provided robust windows for detecting full and partial receptor agonism. In total, the frozen cells were validated for kinase signaling assays for 66 different GPCRs. This research demonstrates that frozen gamma-irradiated cells are a useful tool for characterizing drug
Tyrosine kinases are enzymes that transfer phosphate groups from ATP to tyrosine residues on proteins. There are two main families of tyrosine kinases: receptor tyrosine kinases and non-receptor tyrosine kinases. Receptor tyrosine kinases are transmembrane receptors that transmit extracellular signals to intracellular pathways. Examples include receptors for epidermal growth factor, insulin, fibroblast growth factors, platelet-derived growth factor, nerve growth factor, ephrins, and vascular endothelial growth factor.
miRNA profiling from blood challenges and recommendations - Download the articleQIAGEN
The discovery of stable miRNA species circulating in blood has led to increased research focus on disease-related variations in serum and plasma miRNA expression and the possibility that such variations could serve as noninvasive biomarkers for disease. Working with serum and plasma miRNA presents various challenges in purification and characterization. In this paper, we outline QIAGEN recommendations for robust purification and quantification, as well as reliable data normalization and analysis.
This document discusses tyrosine kinase inhibitors, which are drugs that target tyrosine kinases. It begins by introducing tyrosine kinases and their role in cell signaling pathways. It then describes several important tyrosine kinase inhibitors, including BCR-ABL inhibitors like imatinib, dasatinib, and nilotinib; EGFR inhibitors like gefitinib and erlotinib; and VEGF inhibitors like sunitinib and sorafenib. For each drug, it provides information on mechanisms of action, pharmacokinetics, dosing, toxicity profiles, and FDA-approved indications. The document concludes by discussing mechanisms of resistance to BCR-ABL kinase inhibitors.
Technical Guide to Qiagen PCR Arrays - Download the GuideQIAGEN
Total RNA discovery with RT2 and miScript PCR Arrays : Explore the RNA universe - Whatever your destination within the RNA universe, QIAGEN will help you get there. The miRNeasy kits deliver pure, high-quality total RNA from a broad range of samples. The RT2 and miScript PCR arrays are a complete solution both for focused analysis of gene and microRNA expression and for validation of microarray and RNA sequencing experiments. Together with the powerful analytics tools of GeneGlobe® and QIAGEN Ingenuity® Pathway Analysis, these products give you a smooth path from your sample to high-quality results.
DNA Analysis - Basic Research : A Case StudyQIAGEN
The document discusses using the QIAxcel system for analyzing DNA fragments from genetic analyses like genotyping and mapping mutant genes. It provides examples of using the QIAxcel to genotype Arabidopsis thaliana plants with CAPS markers for a pks3 mutant allele. The system allows accurate sizing of DNA fragments in a high-throughput manner to unambiguously identify genotypes. It also discusses using the QIAxcel to analyze alternative splicing patterns regulated by the Tra2β protein to identify roles of its domains. The system provides rapid, sensitive and reproducible analysis of splicing patterns.
Imatinib is the first FDA approved targeted protein kinase inhibitor for chronic myelogenous leukemia and gastrointestinal stromal tumors. It binds to and inactivates the BCR-ABL tyrosine kinase. Dasatinib and nilotinib are also BCR-ABL tyrosine kinase inhibitors approved for CML. Gefitinib, erlotinib and lapatinib inhibit the epidermal growth factor receptor tyrosine kinase and are approved for various cancers. Sunitinib, sorafenib, pazopanib, and vatalanib inhibit vascular endothelial growth factor receptors and are approved for renal cell carcinoma and other cancers. These oral tyrosine kinase inhibitors are important targeted cancer therapies.
Maximizing PCR and RT-PCR Success - Download the BrochureQIAGEN
The invention of the polymerase chain reaction (PCR) by K. Mullis and coworkers in 1985 revolutionized molecular biology and molecular medicine. Major research areas, such as biomarker discovery, gene regulation and cancer research are challenging today’s PCR technologies with more demanding requirements. These include the need for increased throughput while reducing costs, higher assay sensitivity and reliable data normalization. Assay development and evaluation, reproducibility of data and time to result are still major problems encountered by researchers.
Meeting today’s challenges in PCR requires advances in all methods of the workflow that starts with sample collection, sample stabilization, and nucleic acid purification, and ends with amplification and detection. The following pages focus on the importance of amplification in meeting these challenges.
This document describes a method for determining the phosphorylation motifs of protein kinases using an arrayed combinatorial peptide library. The procedure involves screening 198 biotinylated peptide substrates in the presence of radiolabeled ATP. The peptides are captured on a streptavidin membrane after phosphorylation, and the membrane is exposed to detect incorporation of radiolabel into each peptide, revealing the kinase's phosphorylation motif. The document provides details on expressing and purifying active kinases, optimizing reaction conditions, performing the peptide array screen in 384-well or 1536-well plates, and analyzing the results to determine the kinase's consensus phosphorylation sequence.
Epidermal growth factor (EGF) is a protein that binds to EGF receptors on epithelial and epidermal cells and initiates the EGF signaling pathway. When EGF binds to EGF receptors, it causes them to dimerize and activate their tyrosine kinase activity, leading to phosphorylation and activation of downstream proteins in the MAPK pathway. Mutations that cause constitutive activation of this pathway can lead to uncontrolled cell growth and cancer. Potential cancer treatments discussed include RGD-based peptides that target the αvβ3 integrin receptor involved in angiogenesis, and a conjugate of low molecular weight heparin and suramin that may inhibit tumor growth by blocking vascular endothelial growth factor (VEGF).
The EpiTect Methyl II PCR Array System allows for the fast and accurate detection of regional DNA methylation levels of multiple genes simultaneously, without the need for bisulfite conversion. Using a simple restriction enzyme digestion followed by real-time PCR, the system can analyze methylation levels of 22 or 94 genes. It provides controls and free data analysis tools. The system is well-suited for biomarker characterization and profiling DNA methylation changes related to disease pathways and processes.
This document discusses orphan G protein-coupled receptors (GPCRs) and efforts to identify endogenous ligands for these receptors. It provides background on the IUPHAR database and criteria for designating pairings between orphan receptors and endogenous ligands. 57 class A, 28 class B, and 6 class C orphan GPCRs currently have no reported pairings. The document calls for further research to validate reported pairings and identify ligands for the remaining orphan GPCRs to advance our understanding of their functions and therapeutic potential.
The CRISPR-Cas9 system demonstrates unparalleled genome editing efficiency in a broad range of species and cell types, but it suffers from concerns related to target specificity. Modified guide RNAs and mutant Cas9 proteins have been developed to reduce off-target editing but, in many cases, the alterations also significantly reduce on-target editing performance. In this presentation, Dr Chris Vakulskas discusses a novel, high-fidelity Cas9 protein that reduces off-target gene editing, while maintaining high on-target activity. Dr Vakulskas presents data from the development of the new Alt-R® S.p. HiFi Cas9 Nuclease 3NLS and describes its usefulness in mitigating unwanted off-target gene editing, without the issues associated with transfection of plasmid DNA.
This document discusses chromogenic substrates, which are peptides linked to a chromophore that are used to detect and measure the activity of proteolytic enzymes like thrombin and factor Xa. Chromogenic substrates are cleaved by these enzymes, releasing the chromophore p-nitroaniline which turns yellow and can be quantified. The document provides examples of specific chromogenic substrates for various enzymes and describes their use in clinical assays to measure coagulation factors, antithrombin, heparin, and activated protein C.
Huntington's disease is a genetic disorder caused by a CAG repeat expansion in the HTT gene on chromosome 4. It is characterized by motor dysfunction and cognitive decline that worsen over time. Symptoms appear when around 30-40 years old but can occur earlier. The mutation results in abnormal huntingtin protein that causes neuronal dysfunction and death through disruption of transcriptional pathways and induction of apoptosis. No cure exists but some treatments can help manage symptoms.
1. INTRODUCTION
2. WHAT IS A RECEPTOR
3. HISTORY
4. CONCEPT OF CELL SIGNALLING
5. RECEPTOR SUPER FAMILIES
6. GPCRs- SIGNAL TRANSDUCTION & ITS SECOND MESSENGERS
Nerve growth factor (NGF) Signalling pathways. SAKEEL AHMED
Nerve growth factor is a small protein that promotes the growth, maintenance, and survival of neurons. It was discovered in 1956 by Rita Levi-Montalcini and Stanley Cohen. NGF signals through two pathways, the TrkA pathway which promotes cell survival and the p75 pathway which can promote both survival and apoptosis. NGF production decreases in conditions like Alzheimer's disease, impairing the ability to form new neural connections.
Dasatinib is a second-generation BCR-ABL tyrosine kinase inhibitor that is more potent than imatinib and effective against some imatinib-resistant mutations. Unlike imatinib, dasatinib interferes with platelet function by targeting Src family kinases, which may contribute to increased bleeding risk in patients. Clinical trials found dasatinib achieved high rates of cytogenetic and molecular response in chronic myeloid leukemia patients, including those resistant to or intolerant of imatinib. However, dasatinib treatment also carried risks of myelosuppression and bleeding that required dose reductions in some cases.
Evaluation of the changes in the gene CYP3A4 expression in HepG2 cells under ...Angela Farngren
This study evaluated the effects of rifampicin and alpha-ketoglutarate on expression of the CYP3A4 gene in HepG2 liver cells. HepG2 cells were treated with either 10mM rifampicin or 4mM alpha-ketoglutarate for 7 days. RNA was extracted on days 0, 3, and 7 and analyzed via qPCR. Rifampicin treatment significantly increased CYP3A4 expression, peaking on day 3 and decreasing slightly by day 7. Alpha-ketoglutarate initially decreased CYP3A4 expression for the first 3 days but then increased expression levels. The study demonstrates that rifampicin and alpha-ket
Gamma-irradiated Frozen Cells Validated for GPCR-mediated Kinase SignalingPerkinElmer, Inc.
This document summarizes research from PerkinElmer and TGR Biosciences on using gamma-irradiated frozen cells for G protein-coupled receptor (GPCR) signaling assays. Specifically, it shows that frozen "cAMPZen" and "AequoZen" cells can be used with AlphaScreen SureFire assays to measure kinase pathways like ERK, Akt, and CREB activation downstream of various GPCRs. The assays performed similarly to cultured cells and provided robust windows for detecting full and partial receptor agonism. In total, the frozen cells were validated for kinase signaling assays for 66 different GPCRs. This research demonstrates that frozen gamma-irradiated cells are a useful tool for characterizing drug
Tyrosine kinases are enzymes that transfer phosphate groups from ATP to tyrosine residues on proteins. There are two main families of tyrosine kinases: receptor tyrosine kinases and non-receptor tyrosine kinases. Receptor tyrosine kinases are transmembrane receptors that transmit extracellular signals to intracellular pathways. Examples include receptors for epidermal growth factor, insulin, fibroblast growth factors, platelet-derived growth factor, nerve growth factor, ephrins, and vascular endothelial growth factor.
miRNA profiling from blood challenges and recommendations - Download the articleQIAGEN
The discovery of stable miRNA species circulating in blood has led to increased research focus on disease-related variations in serum and plasma miRNA expression and the possibility that such variations could serve as noninvasive biomarkers for disease. Working with serum and plasma miRNA presents various challenges in purification and characterization. In this paper, we outline QIAGEN recommendations for robust purification and quantification, as well as reliable data normalization and analysis.
This document discusses tyrosine kinase inhibitors, which are drugs that target tyrosine kinases. It begins by introducing tyrosine kinases and their role in cell signaling pathways. It then describes several important tyrosine kinase inhibitors, including BCR-ABL inhibitors like imatinib, dasatinib, and nilotinib; EGFR inhibitors like gefitinib and erlotinib; and VEGF inhibitors like sunitinib and sorafenib. For each drug, it provides information on mechanisms of action, pharmacokinetics, dosing, toxicity profiles, and FDA-approved indications. The document concludes by discussing mechanisms of resistance to BCR-ABL kinase inhibitors.
Technical Guide to Qiagen PCR Arrays - Download the GuideQIAGEN
Total RNA discovery with RT2 and miScript PCR Arrays : Explore the RNA universe - Whatever your destination within the RNA universe, QIAGEN will help you get there. The miRNeasy kits deliver pure, high-quality total RNA from a broad range of samples. The RT2 and miScript PCR arrays are a complete solution both for focused analysis of gene and microRNA expression and for validation of microarray and RNA sequencing experiments. Together with the powerful analytics tools of GeneGlobe® and QIAGEN Ingenuity® Pathway Analysis, these products give you a smooth path from your sample to high-quality results.
DNA Analysis - Basic Research : A Case StudyQIAGEN
The document discusses using the QIAxcel system for analyzing DNA fragments from genetic analyses like genotyping and mapping mutant genes. It provides examples of using the QIAxcel to genotype Arabidopsis thaliana plants with CAPS markers for a pks3 mutant allele. The system allows accurate sizing of DNA fragments in a high-throughput manner to unambiguously identify genotypes. It also discusses using the QIAxcel to analyze alternative splicing patterns regulated by the Tra2β protein to identify roles of its domains. The system provides rapid, sensitive and reproducible analysis of splicing patterns.
Imatinib is the first FDA approved targeted protein kinase inhibitor for chronic myelogenous leukemia and gastrointestinal stromal tumors. It binds to and inactivates the BCR-ABL tyrosine kinase. Dasatinib and nilotinib are also BCR-ABL tyrosine kinase inhibitors approved for CML. Gefitinib, erlotinib and lapatinib inhibit the epidermal growth factor receptor tyrosine kinase and are approved for various cancers. Sunitinib, sorafenib, pazopanib, and vatalanib inhibit vascular endothelial growth factor receptors and are approved for renal cell carcinoma and other cancers. These oral tyrosine kinase inhibitors are important targeted cancer therapies.
Maximizing PCR and RT-PCR Success - Download the BrochureQIAGEN
The invention of the polymerase chain reaction (PCR) by K. Mullis and coworkers in 1985 revolutionized molecular biology and molecular medicine. Major research areas, such as biomarker discovery, gene regulation and cancer research are challenging today’s PCR technologies with more demanding requirements. These include the need for increased throughput while reducing costs, higher assay sensitivity and reliable data normalization. Assay development and evaluation, reproducibility of data and time to result are still major problems encountered by researchers.
Meeting today’s challenges in PCR requires advances in all methods of the workflow that starts with sample collection, sample stabilization, and nucleic acid purification, and ends with amplification and detection. The following pages focus on the importance of amplification in meeting these challenges.
This document describes a method for determining the phosphorylation motifs of protein kinases using an arrayed combinatorial peptide library. The procedure involves screening 198 biotinylated peptide substrates in the presence of radiolabeled ATP. The peptides are captured on a streptavidin membrane after phosphorylation, and the membrane is exposed to detect incorporation of radiolabel into each peptide, revealing the kinase's phosphorylation motif. The document provides details on expressing and purifying active kinases, optimizing reaction conditions, performing the peptide array screen in 384-well or 1536-well plates, and analyzing the results to determine the kinase's consensus phosphorylation sequence.
Epidermal growth factor (EGF) is a protein that binds to EGF receptors on epithelial and epidermal cells and initiates the EGF signaling pathway. When EGF binds to EGF receptors, it causes them to dimerize and activate their tyrosine kinase activity, leading to phosphorylation and activation of downstream proteins in the MAPK pathway. Mutations that cause constitutive activation of this pathway can lead to uncontrolled cell growth and cancer. Potential cancer treatments discussed include RGD-based peptides that target the αvβ3 integrin receptor involved in angiogenesis, and a conjugate of low molecular weight heparin and suramin that may inhibit tumor growth by blocking vascular endothelial growth factor (VEGF).
The EpiTect Methyl II PCR Array System allows for the fast and accurate detection of regional DNA methylation levels of multiple genes simultaneously, without the need for bisulfite conversion. Using a simple restriction enzyme digestion followed by real-time PCR, the system can analyze methylation levels of 22 or 94 genes. It provides controls and free data analysis tools. The system is well-suited for biomarker characterization and profiling DNA methylation changes related to disease pathways and processes.
This document discusses orphan G protein-coupled receptors (GPCRs) and efforts to identify endogenous ligands for these receptors. It provides background on the IUPHAR database and criteria for designating pairings between orphan receptors and endogenous ligands. 57 class A, 28 class B, and 6 class C orphan GPCRs currently have no reported pairings. The document calls for further research to validate reported pairings and identify ligands for the remaining orphan GPCRs to advance our understanding of their functions and therapeutic potential.
The CRISPR-Cas9 system demonstrates unparalleled genome editing efficiency in a broad range of species and cell types, but it suffers from concerns related to target specificity. Modified guide RNAs and mutant Cas9 proteins have been developed to reduce off-target editing but, in many cases, the alterations also significantly reduce on-target editing performance. In this presentation, Dr Chris Vakulskas discusses a novel, high-fidelity Cas9 protein that reduces off-target gene editing, while maintaining high on-target activity. Dr Vakulskas presents data from the development of the new Alt-R® S.p. HiFi Cas9 Nuclease 3NLS and describes its usefulness in mitigating unwanted off-target gene editing, without the issues associated with transfection of plasmid DNA.
This document discusses chromogenic substrates, which are peptides linked to a chromophore that are used to detect and measure the activity of proteolytic enzymes like thrombin and factor Xa. Chromogenic substrates are cleaved by these enzymes, releasing the chromophore p-nitroaniline which turns yellow and can be quantified. The document provides examples of specific chromogenic substrates for various enzymes and describes their use in clinical assays to measure coagulation factors, antithrombin, heparin, and activated protein C.
Huntington's disease is a genetic disorder caused by a CAG repeat expansion in the HTT gene on chromosome 4. It is characterized by motor dysfunction and cognitive decline that worsen over time. Symptoms appear when around 30-40 years old but can occur earlier. The mutation results in abnormal huntingtin protein that causes neuronal dysfunction and death through disruption of transcriptional pathways and induction of apoptosis. No cure exists but some treatments can help manage symptoms.
1. INTRODUCTION
2. WHAT IS A RECEPTOR
3. HISTORY
4. CONCEPT OF CELL SIGNALLING
5. RECEPTOR SUPER FAMILIES
6. GPCRs- SIGNAL TRANSDUCTION & ITS SECOND MESSENGERS
Nerve growth factor (NGF) Signalling pathways. SAKEEL AHMED
Nerve growth factor is a small protein that promotes the growth, maintenance, and survival of neurons. It was discovered in 1956 by Rita Levi-Montalcini and Stanley Cohen. NGF signals through two pathways, the TrkA pathway which promotes cell survival and the p75 pathway which can promote both survival and apoptosis. NGF production decreases in conditions like Alzheimer's disease, impairing the ability to form new neural connections.
Basic Mutagenic signal Transduction or the cancer signal transduction that control cell cycle are important pathways to understand cancer in molecular level and to invent targeted treatment.
This document summarizes protein kinases and kinase recognition sites. It discusses that protein kinases modify other proteins by attaching phosphate groups and are involved in key cellular processes like glucose metabolism and cell proliferation. It then describes various types of kinases like PKA, PKC, PDK1 and their functions. The document further classifies kinases and discusses kinase recognition motifs. Finally, it summarizes different types of crosstalk between kinases, such as arginine methylation blocking phosphorylation and vice versa, as well as distal crosstalk impacts.
This document discusses tyrosine kinase inhibitors and their role in cancer therapy. It begins by introducing tyrosine kinases and their importance in cellular signaling pathways. Tyrosine kinases are implicated in cancer development and progression. The document then discusses the classification, structure, and mechanisms of tyrosine kinase receptors. It provides examples of FDA-approved tyrosine kinase inhibitors for various cancers. The document discusses strategies for inhibiting EGFR signaling, including monoclonal antibodies and small molecule tyrosine kinase inhibitors. It also provides information on trastuzumab and its role and use for HER2-positive breast cancer.
The document discusses three main types of receptors: ligand-gated receptors, enzyme-linked receptors, and nuclear receptors. Ligand-gated receptors include nicotinic acetylcholine receptors and GABAA receptors, which act as ion channels and mediate fast synaptic transmission. Enzyme-linked receptors include tyrosine kinase receptors, JAK/STAT receptors, Toll-like receptors, and guanylyl cyclase receptors, which activate intracellular enzyme pathways to regulate processes like cell growth and inflammation. Nuclear receptors directly bind to DNA and act as transcription factors to regulate gene expression, responding to ligands like steroids, vitamins, and fatty acids.
Metabolic investigation of segmental overgrowth: new insights in pathogenic m...BiologInc
This document summarizes a study investigating the metabolic mechanisms underlying segmental overgrowth disorders and potential treatments. The study found that mutations in the PI3K-AKT pathway, such as in PIK3CA and AKT1, lead to increased cell proliferation and metabolism. Functional studies showed that cell lines with PIK3CA mutations had increased AKT phosphorylation and responded strongly to growth factors, while AKT1 mutant cell lines responded less. Treatment with PI3K or mTOR inhibitors normalized the metabolic profile in both AKT1 and PIK3CA mutant cell lines, suggesting these may be effective treatments. The study provides new insights into the molecular mechanisms and potential targeted therapies for segmental overgrowth disorders.
The PI3K-Akt-mTOR pathway is an intracellular signal transduction pathway that promotes metabolism, proliferation, cell survival, growth and angiogenesis. Key components include receptor tyrosine kinases, PI3K, PIP2, PIP3, and Akt. Akt is activated by phosphorylation and regulates various proteins involved in functions like cell growth. Dysregulation of this pathway can lead to cancer due to abnormal cell proliferation and is associated with neurodevelopmental disorders.
1) LAR is a receptor for chondroitin sulfate proteoglycans (CSPGs), which are inhibitory to axon growth in the central nervous system.
2) CSPGs bind to LAR with high affinity and stimulate its phosphatase activity. Blocking LAR reverses CSPG-mediated growth inhibition.
3) In mouse models of spinal cord injury, peptides targeting LAR promote axon growth, functional recovery, and behavioral improvements by overcoming the inhibitory effects of CSPGs. LAR blockade may be a promising therapeutic approach for central nervous system axon injury.
The document summarizes the mechanisms and differences between RNA interference (RNAi) and RNA activation (RNAa). RNAi involves short-term gene silencing through mRNA cleavage or translational repression in the cytoplasm, mediated by Argonaute 2. RNAa induces long-term gene activation through epigenetic changes and transcriptional activation in the nucleus. While RNAi has been well characterized at the molecular level, the mechanism of RNAa is not fully understood. RNAa has been shown to activate various tumor suppressor and stem cell genes in several human and animal cell lines.
This document provides an overview of the topics covered in an advanced cell biology course, including cell signaling pathways. The course covers cell structures like the nucleus and membranes, as well as processes like transcription, splicing, trafficking and the cell cycle. It focuses on how cells use receptor proteins and second messengers to transduce chemical and physical stimuli into responses that regulate gene expression, metabolism, secretion and motility. Specific signaling pathways discussed include G protein-coupled receptors, receptor tyrosine kinases, cytokine receptors, MAP kinase pathways, insulin signaling, TGF-beta signaling, calcium signaling and visual transduction in vertebrates.
Tyrosine kinases are enzymes that mediate cell signaling pathways involved in processes like proliferation, differentiation, and apoptosis. They are classified as receptor tyrosine kinases, which are transmembrane proteins, or non-receptor tyrosine kinases, which are cytoplasmic. Oncogenic activation of tyrosine kinases can occur through autocrine activation, mutations, rearrangements or amplifications. Tyrosine kinases are targets for anticancer agents, including small molecule inhibitors that bind the ATP site, monoclonal antibodies, and inhibitors of heat shock proteins. Tyrosine kinase inhibitors include Gleevec, Iressa, and Tarceva.
This document discusses surface modification of nanoparticles for biomedical applications. It describes how nanoparticles can be modified on their surface with various ligands to target specific cells and tissues. These ligands include antibodies, peptides, aptamers, and other molecules like folate. Aptamers and peptides offer advantages over antibodies as targeting ligands. The document provides examples of using various ligands like VEGF, folate, and transferrin to target receptors overexpressed on cancers and other diseases. It also discusses different conjugation chemistries used to attach ligands to the nanoparticle surface, like using succinimide, biotin-streptavidin, and thiol chemistry.
Protein kinases are the most important biochemical regulatory system in animal cells and comprise about 1.7% of the human genome. They are classified into major groups including serine/threonine kinases, tyrosine kinases, dual specificity kinases, and atypical protein kinases. Key protein kinases include PKA, PKG, PKC, calcium/calmodulin dependent kinases, MAP kinases, and receptor and non-receptor tyrosine kinases. Dysregulation of protein kinases contributes to diseases like cancer, and many cancer therapies target abnormal protein kinase signaling pathways.
Gentile Alessandra Torino 13° Convegno Patologia Immune E Malattie Orfane 21 ...cmid
The document discusses the MET oncogene and its role in cancer progression and invasion. It describes how the MET oncogene encodes the HGF receptor and regulates genes involved in invasive growth. The MET oncogene can become activated in cancer through overexpression, mutations, or autocrine loops, making it reliant on continued activation. Therapeutic strategies aim to inhibit the MET tyrosine kinase through small molecule inhibitors or antibodies to treat cancers dependent on MET signaling. Dependence on the MET oncogene often correlates with its amplification in cancer tumors.
The document summarizes a study analyzing a single nucleotide polymorphism (SNP) in the actin filament associated protein (AFAP-110) gene in patients with head and neck cancer. The SNP leads to an amino acid change that enhances activation of c-Src kinase, which promotes tumor growth. The study found that all 50 patients had the polymorphic AFAP-110403C genotype, with 13 patients being heterozygous and 37 being homozygous. Further analysis is underway to determine if the AFAP-110 SNP correlates with tumor measurements, biomarkers of response, and clinical outcomes in patients treated with dasatinib, a Src inhibitor, and erlotinib, an EGFR inhibitor.
Rho GTPases as regulators of morphological neuroplasticityFatih University
Rho GTPases play a key role in regulating neuronal morphology by mediating interactions between cell adhesion molecules and the cytoskeleton. Specifically, Rho inhibits neurite extension while Rac and Cdc42 promote neurite outgrowth and dendritic spine formation. In glial cells, Rho GTPases are involved in myelination by oligodendrocytes and Schwann cells. The role of RhoA in particular contributes to the inhibitory environment of the CNS that prevents axon regeneration.
This document summarizes various targeted anticancer therapies. It discusses targeted therapies that interfere with molecular structures implicated in tumor growth like nuclear factors, cell survival factors, and angiogenesis factors. Primary targeted therapy tools are monoclonal antibodies and small synthetic molecules. Protein kinases and their role in signaling pathways are described. Examples of targeted therapies discussed include BCR-ABL tyrosine kinase inhibitors, EGFR inhibitors, HER2/NEU inhibitors, angiogenesis inhibitors targeting VEGF, mTOR inhibitors, proteasome inhibitors, MAPK pathway inhibitors, and monoclonal antibodies. Resistance mechanisms and newer agents to overcome resistance are also summarized.
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2. Kinases and Drug Discovery
• 518 Kinases in human genome
• 214 Kinases implicated in disease
• >30% of drug discovery programs
target kinases
• 240 compounds targeting protein
kinases were in development in
05/2004
– 145 in preclinical development
– 27 in PI
– 45 in PII
– 24 in PIII
• Compounds in clinical trials target
about 20 different kinases
– Oncology focused
Manning et al., Science, 6 December 2002
2
3. Kinases Are Validated Therapeutic Targets
Product Company Kinase Target(s) Approved Indications
Gleevec§ BCR/ABL, PDGFR, CML, gastrotintestinal stromal
Novartis
(imatinib) KIT tumors
Nexavar§ Raf, VEGFR-2,
Bayer/Onyx VEGFR-3, KIT, FLT- renal cell carcinoma
(sorafenib) 3, PDGFR-ß
Sutent VEGFR, PDGFR, renal cell carcinoma,
Pfizer
(sunitinib) KIT, FLT-3 gastrointestinal stromal tumors
Tarceva OSI/Genentech/
EGFR NSCLC, pancreatic cancer
(erlotinib) Roche
Iressa
AstraZeneca EGFR NSCLC
(gefitinib)
Sprycel
BMS BCR/ABL, SRC CML, ALL
(dasatinib)
Eril cerebral vasospasm resulting from
Asahi Kisei ROCK
(fasudil) subarachnoid hemorrhage (Japan)
§Binds to the inactive, “DFG-out” conformation of the target kinase(s)
3
4. p38 MAP Kinase as a Drug Target
• MAP kinases integrate, process large number of
extracellular signals
• 3 distinct MAPK pathways
– ERK
• Activated by mitogenic, proliferative stimuli
– JNK
– p38
• Both activated by environmental stress
– Includes inflammatory cytokines
– 60-70% identical
• Differ mainly in sequence, size of activation loop
4
5. Regulation of Cellular Responses by p38
• p38 regulates gene transcription by direct
phosphorylation of transcription factors
• p38 regulates mRNA stability by activating downstream
kinases
– Phosphorylation of AU-rich binding proteins stabilizes IL-1, COX-
2, other inflammatory transcripts
• p38 regulates mRNA translation by activating
downstream kinases
– Translational control proteins
– Major mechanism of p38 effects on TNF
• p38 regulates histone 113 phosphorylation
– NF-kB binding sites upstream of IL-8, MCP-1, other genes
accessible
5
6. p38 Inhibition as a Strategy to Attack Chronic
Inflammatory States
LPS
IL-1β
β
TNFαα
MKK3
MKK6
P38 Kinase
p38 Inhibitors
Inflammation
MAPKAP K2
TRANSLATIONAL
REPRESSION RELEASE
IL-1β
β
Pre-IL-1β
β TNFαα
Pre-TNFαα
6
7. Rationale for p38 Inhibitors in Treatment of RA
and Other Diseases
• p38 regulates cytokine production at transcriptional and
translational levels
• p38 regulates chemotaxis at level of chemokine
expression and cellular chemotactic response
• Variety of chemotypes active in various preclinical
models
– AA and CIA in rodents
– Streptococcal cell wall-induced arthritis
– LPS challenge
– Ischemia/reperfusion in heart, liver, lung
– Cardiac hypertrophy
• Anti-TNF and anti-IL-1 biologics’ efficacy in RA, psoriasis,
Crohn’s disease
7
8. p38 Inhibitors Discontinued From Clinical
Development
• VX745
– 12 weeks 250 mg BID
– ACR20 benefits
– Liver enzyme elevations, other signs
– CNS effects in dog reported
• BIRB796
– Elevated liver enzymes reported in Phase 1 studies
– ~2 uM EC50 in ex vivo LPS challenge
– Reported no efficacy in Crohn’s trial
• RO-3201195
– 75% inhibition of ex vivo LPS-induced IL-1β production by
750 mg BID in 28 day study
8
9. p38 Inhibitors in Clinical Development
• Previous molecules have been dose-limited
by adverse events
– LFT abnormalities
– Rash
– GI irritation
– CNS toxicity
– QTc prolongation
• Lack of unifying toxicity implies chemotype
rather than target
• Strategies that increase selectivity to target
may increases chances of clinical success
9
10. p38 Inhibitors in Clinical Development
• Hypothesis: “safe enough” p38 inhibitor will be medically useful in
RA and other autoimmune/inflammatory conditions driven by IL-1β,
TNFα
• Publicly available data from Vertex in 12 week RA studies
ACR20
10mg VX702 5mg VX702 placebo
40% 38% 30%
250 mg bid VX745 placebo
43% 17%
10
11. Kemia’s Approach To The Challenges in
Kinase Drug Discovery
• Challenges
– Crowded chemical intellectual property space focusing on ATP-
competitive scaffolds
– Poor selectivity of inhibitors
– Clinical toxicities
• Kemia’s Approach
– Target novel chemical space distant from the typical “purine-like”
chemistries
– Target inactive kinase conformations that are incompatible with
ATP-binding
– Utilize slow off-rates to optimize PK/PD relationships that
increase therapeutic indices.
11
12. Many Kinases are Potential Targets for DFG-Out
Inhibitors
• Crystal structures with the inactive DFG-out
conformation have been solved for several kinases
– Tyr kinases - INSR, VEGFR-2, Tie-2, MUSK, IGF1R, ABL,
SRC, FLT3
– Ser/Thr kinases - PKB, Akt-2, p38, RAF
• Additional kinases have the potential to adopt the DFG-
out conformation
• Multiple examples of inhibitors targeting the DFG-out
domain (Gleevec, Nexavar, etc.) provide a motivation
for designing inhibitors targeting kinases of therapeutic
interest
12
13. DFG-In Versus DFG-Out Kinase Inhibitors
• Type I Inhibitors
• Bind in the region normally occupied by the adenine ring of ATP and make similar
contacts to the “hinge” region
• Bind ubiquitous sites that make the design of highly selective inhibitors
problematic
• Bind to the “active” conformation of the kinase similar to that seen with ATP
bound
• Represent the majority of programs that have targeted protein kinases (crowded
IP space)
• Type II Inhibitors
• Bind to regions adjacent to the ATP binding site although can make contacts to
the “hinge” region
• Bind sites that contain significant structural variation that allow for the design of
highly selective inhibitors
• Bind to and stabilize an “inactive” conformation of the kinase with a distinct
(“DFG-out” or “Phe-out”) conformation of the activation loop
• In some cases have very slow off rates (long duration of action)
• Represent minority kinase drug discovery programs to date (greater freedom to
operate)
13
14. Kémia’s Chemistries
• >3000 compounds have been designed and synthesized as
Type II binders for kinases
– Represent several chemical scaffolds
• Chemical scaffolds have been optimized to limit DMPK or
toxicity liabilities
– Solubility
– PAMPA, CACO2
– HLM stability
– Plasma stability
– Plasma protein binding
– CYP inhibition
– hERG inhibition
• Strong intellectual property position
14
15. Targeting p38 for Inhibition
• Publicly available co-crystal structures
– DFG-in
– DFG-out
• Molecular Modeling
• Conventional moieties tied together by a
variety of cores to target
– DFG-out pocket
– Specificity pocket
– Hinge region
15
16. KC706 Summary of In Vitro Results
• Potent, selective p38α inhibitor targeting the Phe-out
pocket
– IC50 = 60 nM (kinase assay)
– IC50 = 50 nM (LPS-stimulated TNFα secretion from THP-1 cells)
– ~10-fold selective vs p38ß, very weak inhibitor of p38γ and
p38δ
– Excellent selectivity profile versus a panel of off-target kinases
• Prevents p38α phosphorylation/activation by upstream
kinases (MKK3/6)
• Slow off-rate/long duration of action (biochemical and
cell-based assays)
• Inhibits LPS-stimulated TNFα and IL-1ß production in
human/rat whole blood
16
17. Time-Dependent Inhibition of p38α Enzymatic
Activity by KC706
100
Preincubation IC50
% Inhibition
75 Time (nM)
0 302
50 t = 0 min 30 38
t = 30 min 60 14
25 t = 60 min 120 8
t = 120 min
0
10 -9 10 -8 10 -7 10 -6 10 -5
[KC706] (M)
Inhibition of enzymatic activity of recombinant human p38α.
Preincubations at 37ºC.
17
18. KC706 Exhibits Time-Dependent IC50 Shift
Characteristic of Some Type II Inhibitors
50 KC706
BIRB796
40 SB-203580
IC50 Ratio*
VX745
30
IC 50 @ 0 min
* IC 50 Ratio =
IC 50 @ time = t
20
10
0
0 50 100
Time
Inhibition of recombinant active p38α
18
19. Type II Inhibitors of p38α Exhibit Long Duration
of Binding
Biacore Analysis
Binding kon koff KD t1/2 Relative
Compound
Mode (M-1sec-1) (sec-1) (nM) (min) Offrate*
SB-203580 Type I 6.1 x 106 0.171 28 <0.1 1
Kémia Series
Type II 0.94 x 104 3.9 x 10-4 41 ~30 438-fold
A Example
Kémia Series
Type II 1.13 x 104 6.2 x 10-4 55 ~19 276-fold
B Example
Studies utilized recombinant, activated p38α at 25°C
*Relative off-rate = t1/2 for indicated compound / t1/2 for SB-203580
19
21. KC706 Inhibition Exhibits Long Duration of Binding,
Stabilization of DFG-out Conformation
• Slow inhibitor binding kinetics
• Indirect evidence for Type II-like mode of
action
– Modeling fits best to DFG-out conformation
– Inhibition of phosphorylation of p38 under
“short” assay conditions
21
22. KC706 Prevents p38α Phosphorylation & Activation
By Upstream Kinases (MKK3/6)
• p38 activated by dual phosphorylation on
Thr180 and Tyr182
• Upstream kinases MKK6 and MKK3
phosphorylate these residues in response to
signals upstream of them
• Phospho-specific antibody detection of
phosphorylation of TGY motif standard method
of detecting p38 activation
• Distinct from MAPKK-independent mechanisms
such as TAB1 and the Lck-ZAP70 mechanism
described by Prof. Miceli
22
23. DFG-Out Inhibitors Function Differently
From ATP-Competitive Inhibitors
Activation PO4 PO4
ATP
Phe Phe Phe
MKK3/MKK6
Inactive protein
“Active” p38
alternates between Phe- ATP-competitive inhibitors
in and Phe-out bind in this conformation
conformations
“Phe-out”
Phe
Inhibitor
PO 4
Phe Phe
“Inactive” p38 Conformation
Phe-out
Inhibitor
p38 locked in Phe-out Phospho-p38 locked in Phe-out
PO4 by MKK3/6 inhibited does not bind ATP
“Inactive” p38 “Inactive” p38
23
24. Targeting Active Versus Inactive Conformations
(DFG-Out) of Kinases
Phe Phe
Activation loop
Activation loop
• Traditional kinase inhibitors (left) compete for binding of ATP to the active conformation
• Allosteric inhibitors (right) stabilize an inactive conformation that cannot bind ATP
24
25. KC706 Inhibits LPS-Induced Phosphorylation
of p38α in Human Whole Blood
100
p38 Phosphorylation
(% Inhibition) 80
60
40
20
0
10 -8 10 -7 10 -6 10 -5
[KC706] (M)
1. Human whole blood pretreated 30 min with KC706
2. Add LPS, incubate 20 min
3. Fix and permeabilize cells
4. Stain with anti-pp38 antibody and control antibodies
5. Flow cytometry
25
26. Selectivity of KC706 Across 45 Kinases (Cerep)
p38α
Potency: Red > Black > Green
BIRB796 KC706 SB203580
BIRB796 KC706 SB203580
26
27. KC706 Inhibits LPS-Induced TNFα Production
in Human Whole Blood
120
KC-706
TNFα Secretion 100 BIRB796
(% Inhibition)
80
60
40
20
0
10 -8 10 -7 10 -6 10 -5
[Compound] (M)
1. Human whole blood diluted 1:1 with RPMI-1640
2. Treat 4 hrs with LPS
3. Quantitate TNFα in supernatant
27
28. KC706 Inhibits LPS-Induced TNFα and IL-1β
Production in Human Whole Blood
TNFα IL-1β
% Inhibition IL-1beta Response
% Inhibition TNF Response
100 100
50 50 IC50 ~70 nM
IC50 ~1300 nM
0 0
-9 -8 -7 -6 -5 -4 -9 -8 -7 -6 -5 -4
Concentration KC706 (logM) Concentration KC706 (logM)
1. Human whole blood diluted 1:1 with RPMI-1640
2. Treat 4 hrs with LPS
3. Quantitate TNFα and IL-1β in supernatant
28
29. KC706 Non-Clinical Pharmacology and
Pharmacokinetics
• Active in acute and sub-chronic models of inflammation
– Carrageenan paw edema (CPE; paw edema and IL-1ß mRNA
induction)
– LPS-stimulated TNFα production
– Collagen induced arthritis (CIA; mice and rats)
• Good pharmacokinetic profile in rats
– Oral bioavailability (%F) ~75 %
– Clearance (Cl) ~19 mL/min/kg
– Terminal half-life (t1/2) ~3-4 hrs
– Volume of distribution (Vss) ~5 L/kg
29
30. Orally Administered KC706 Reduces LPS-
Induced TNFα Levels In Vivo
N=10
6000
5000
pg/ml TNFα
4000
3000
N=6
2000
1000
N=10
0
S
PS
e
in
LP
/L
al
/
6*
le
/s
c
e
70
hi
cl
C
hi
ve
K
ve
In vivo LPS challenge in rat
*30 mg/kg PO
30
31. KC706 Reduces Carrageenan-Induced IL-1ß
mRNA Induction In Vivo
400 No Carrageenan
(Arbitrary Units) Carrageenan
IL-1β mRNA
300
200
100
0
e
)
kg
cl
hi
g/
Ve
0m
(3
6
Rats given vehicle or KC706 PO at t = -2 hrs
70
C
Carrageenan injection at t = 0
K
Sacrifice and isolate total RNA from paw at t = 6 hrs
Quantitative RT-PCR
31
32. Acute Anti-inflammatory Efficacy in Rat
Carrageenan-Induced Paw Edema by KC706
EFFECT OF ORALLY ADMINISTERED KR-002524 ON CARRAGEENAN-INDUCED PAW
EDEMA IN RATS
2.00
1.80
CHANGE IN PAW VOL. (ml)
1.60
1.40
1.20
1.00
0.80
0.60
0.40
0.20 Vehicle 3m g/kg 10m g/kg 30m g/kg Indom ethacin
0.00
0 2 4 6
TIME (hr)
Rats given vehicle or KC706 PO at t = -2 hrs
Carrageenan injection at t = 0
N = 6 animals/group
32
33. Dose-Dependent Reversal of Signs of
Collagen-Induced Arthritis by KC706
Ankle Diame ter Ove r T ime - KC706
* p ≤0.05 t-test to Arthritis+Vehicle
0.3 45 Normal + V ehicle
Arthritis + V ehicle
KR -00 252 4 30 mg/kg
0.3 35 KR -00 252 4 8 mg/kg
KC706
KR -00 252 4 2 mg/kg
0.3 25 KR -00 252 4 0.4 mg/kg
D ex 0.0 75 mg/kg
0.3 15 E nbrel 10 mg/kg
0.3 05 Bolder BioPATH, Inc.
N=4 rats: Normal Controls
0.2 95 * * *
N=8 rats/treatment group
* * *
* *
0.2 85
* * *
0.2 75 *
* * *
* * * *
0.2 65 *
0.2 55 * 2 * * * * *
Day 0 *
Day 1 *
Day *
Day 3 *
Day 4 *
Day 5 *
Day 6 Day 7
*
Study Day
33
34. KC706 in Clinical Trials
• Initial Phase I trials have been completed
– Highest dose in excess of expected therapeutic
dose
– Excellent bioavailability and dose proportionality
– No drug-related adverse events
– No liver toxicities observed
– Minimal food effect (top single dose)
– Unconjugated bilirubin elevations from partial
UGT1A1 inhibition guided Phase II dosing to
300mg and below
34
35. KC706 Phase 1 Ex Vivo LPS Challenge
Confirms Anti-inflammatory Effect in Man
• Ex vivo LPS challenge assays anti-inflammatory
effect on peripheral blood cells
– Blood sample before and after drug administration
– Blood samples exposed to LPS (bacterial toxin)
– Immune response measured by IL-1ß, TNFα, or
other marker
– Effect assayed by comparing LPS-stimulated
inflammatory cytokines from pre- and post-drug
blood samples
35
37. KC706 Inhibition of IL-1β Response to Ex Vivo
LPS Challenge: Long Duration of Action
320 mg Day 12
250
% Baseline IL-1b Response
200 *
150
*
100 *
50
* % Baseline normalized
0
placebo 1hr 6hr 12 hr 24hr
Time
* Statistically significant effect (p <0.05)
37
38. KC706 Current Status
• Challenges in kinase drug discovery
– Crowded chemical intellectual property space focusing on ATP-
competitive scaffolds
– Poor selectivity of inhibitors
– Clinical toxicities
• Kémia’s Approach
– Target novel chemical space distant from the typical “purine-like”
chemistries
– Target kinase conformations that minimize ATP binding
– Utilize slow off-rates to optimize PK/PD relationships that
increase therapeutic indices
• Phase 2a trials with KC706 in RA, Dyslipidemia and
Pemphigus Vulgaris
38
39. p38 Team
• Chemistry
– Antonio Garrido Montalban, Eddine Saiah, Erik Boman, Susana
Conde Ceide, Russell Dahl, David Dalesandro, Nancy G.J.
Delaet, Eric Erb, Justin Ernst, Jeff Kahl, Hiroshi Nakanishi, Ed
Roberts, Robert Sullivan, Zhijun Wang, Nathan Kroll
• Biology
– Stephen G. Miller, Christopher J. Larson, Linda Kessler, Andrew
Gibbs, Jeff Kucharski
• Pharmacology
– Jan Lundstrom, Alison Bendele, Phil Bendele, Sean O’Neill,
Valerie Lowe
• ADMET
– Chau-Dung Chang, Marianne Quintos, Barbara Winningham,
Arnie Garcia, Pauline Chai
• Clinical Development
– Bernard D. King, Constance Crowley, David Shapiro, Bonnie
Hepburn
39