This document discusses opportunities for developing novel compounds targeting medically relevant protein families using non-mainstream scaffolds. It notes 400 novel scaffolds in a European compound library that exhibit structural complexity, stereochemistry, scaffold diversity, and challenging chemistry. The document discusses opportunities for targeting G protein-coupled receptors, kinases, and protein-protein interactions using spirocyclic, anellated, and DNA-encoded scaffold libraries. It emphasizes the potential of these approaches for developing compounds with improved target residence time and selectivity profiles through disrupting a protein's hydrophobic spine formation.
Lessons learned from high throughput CRISPR targeting in human cell linesChris Thorne
In just a short period of time CRISPR-Cas9 technology has revolutionized the field of genome editing, and taken the scientific community by storm. Already our understanding of how best to apply this technology has advanced significantly and almost every week new publications appear showcasing its application in basic and translational research.
While CRISPR-Cas9 is applicable across many different cell types, we have found it particularly suited for genome editing in near-haploid human cell lines. This has allowed us to establish a robust pipeline for the inactivation of non-essential genes at unprecedented scale and efficiency.
We have now knocked out over 1500 human genes and have generated a resource that is, to the best of our knowledge, the largest collection of human knockout cell lines available, covering comprehensive subsets of genes clustered by biological pathway (e.g. the autophagy pathway, the JAK/STAT pathway) or by phylogenetic relationship (e.g. kinases, bromodomain-containing proteins).
In this talk we will discuss how, through more than 1500 genome editing experiments, we have started to unravel some of the general principles governing the use of CRISPR-Cas9 in mammalian cells. For example, we have analyzed the impact of variation in the guide RNA sequence on Cas9 cleavage efficiency and characterized the mutational signature arising from CRISPR-Cas9 cleavage.
We will also highlight (with examples) how these learnings are now being applied to introduce other genomic modifications in a high throughput manner, including chromosomal deletions, translocations, point mutations and endogenous gene tags.
Speaker: Benedict C. S. Cross, PhD, Team leader (Discovery Screening), Horizon Discovery
CRISPR–Cas9 mediated genome editing provides a highly efficient way to probe gene function. Using this technology, thousands of genes can be knocked out and their function assessed in a single experiment. We have conducted over 150 of these complex and powerful screens and will use our experience to guide you through the process of screen design, performance and analysis.
We'll be discussing:
• How to use CRISPR screening for target ID and validation, understanding drug MOA and patient stratification
• The screen design, quality control and how to evaluate success of your screening program
• Horizon’s latest developments to the platform
• Horizon’s novel approaches to target validation screening
GENESIS™: Comprehensive genome editing - Translating genetic information into personalised medicines.
Horizon is the only source of rAAV expertise and is uniquely capable of exploiting multiple platforms: CRISPR, ZFNs and rAAV singularly or combined. Horizon’s scientists are experts at all forms of gene editing and so have the experience to help guide customers towards the approach that best suits their project
How to Standardise and Assemble Raw Data into Sequences: What Does it Mean fo...Joseph Hughes
11th OIE Seminar at the XVII INTERNATIONAL SYMPOSIUM OF THE WORLD ASSOCIATION OF VETERINARY LABORATORY DIAGNOSTICIANS (WAVLD)
Saskatoon - 17th June 2015
Recent breakthroughs in genome editing technology have led to a rapid adoption that parallels that seen with RNAi. And like RNAi, these methods are taking the scientific world by storm, with high profile publications in fields as diverse as HIV treatment, stem cell therapy, food crop modification and drug development to name but a few.
Critically, the endogenous modification of genes enables the study of their function in a physiological context. It also overcomes some of the artefacts that can result from established techniques such as transgenesis and RNAi, which have mislead researchers with false positives or negatives. Until recently however genome editing required considerable technical expertise, and consequently was a relatively niche pursuit.
In this talk we will look at how the latest developments in genome editing tools have changed this, with improvements in both ease-of-use and targeting efficiency, as well as a concomitant reduction in costs opening up these approaches to the wider scientific community.
Rapid adoption of the CRISPR/Cas9 system has for example led to a long list of organisms and tissues in which genetic changes have been made with high efficiency. Other technologies such as recombinant adeno-associated virus (rAAV) offer further precision, stimulating the cell’s high-fidelity DNA repair pathways to insert exogenous sequence with unrivalled specificity. Targeting efficiency can be improved still further by using the technologies in combination – genome cutting induced by CRISPR can significantly enhance homologous recombination mediated by rAAV.
Despite these rapid advances, some pitfalls remain, and so we’ll discuss some of the key considerations for avoiding these, ranging from simply picking the right tool for the job to designing an experiment that maximises chances of success.
Finally we’ll look at how genome editing is being applied to both basic and translational research, and in both a gene-specific and genome wide manner. For the study of disease associated genes and mutations scientists can now complement wide panels of tumour cells with genetically defined isogenic cell pairs identical in all but precise modifications in their gene of interest. The ease-of-design and efficiency of the CRISPR system is also being exploited for genome wide synthetic lethality screens, facilitating rapid drug target identification with significantly reduced risk of false negatives and off-target false positives. And again, further synergies are achieved when these approaches are combined to look for potential synthetic lethal targets in specific genomic contexts.
Lessons learned from high throughput CRISPR targeting in human cell linesChris Thorne
In just a short period of time CRISPR-Cas9 technology has revolutionized the field of genome editing, and taken the scientific community by storm. Already our understanding of how best to apply this technology has advanced significantly and almost every week new publications appear showcasing its application in basic and translational research.
While CRISPR-Cas9 is applicable across many different cell types, we have found it particularly suited for genome editing in near-haploid human cell lines. This has allowed us to establish a robust pipeline for the inactivation of non-essential genes at unprecedented scale and efficiency.
We have now knocked out over 1500 human genes and have generated a resource that is, to the best of our knowledge, the largest collection of human knockout cell lines available, covering comprehensive subsets of genes clustered by biological pathway (e.g. the autophagy pathway, the JAK/STAT pathway) or by phylogenetic relationship (e.g. kinases, bromodomain-containing proteins).
In this talk we will discuss how, through more than 1500 genome editing experiments, we have started to unravel some of the general principles governing the use of CRISPR-Cas9 in mammalian cells. For example, we have analyzed the impact of variation in the guide RNA sequence on Cas9 cleavage efficiency and characterized the mutational signature arising from CRISPR-Cas9 cleavage.
We will also highlight (with examples) how these learnings are now being applied to introduce other genomic modifications in a high throughput manner, including chromosomal deletions, translocations, point mutations and endogenous gene tags.
Speaker: Benedict C. S. Cross, PhD, Team leader (Discovery Screening), Horizon Discovery
CRISPR–Cas9 mediated genome editing provides a highly efficient way to probe gene function. Using this technology, thousands of genes can be knocked out and their function assessed in a single experiment. We have conducted over 150 of these complex and powerful screens and will use our experience to guide you through the process of screen design, performance and analysis.
We'll be discussing:
• How to use CRISPR screening for target ID and validation, understanding drug MOA and patient stratification
• The screen design, quality control and how to evaluate success of your screening program
• Horizon’s latest developments to the platform
• Horizon’s novel approaches to target validation screening
GENESIS™: Comprehensive genome editing - Translating genetic information into personalised medicines.
Horizon is the only source of rAAV expertise and is uniquely capable of exploiting multiple platforms: CRISPR, ZFNs and rAAV singularly or combined. Horizon’s scientists are experts at all forms of gene editing and so have the experience to help guide customers towards the approach that best suits their project
How to Standardise and Assemble Raw Data into Sequences: What Does it Mean fo...Joseph Hughes
11th OIE Seminar at the XVII INTERNATIONAL SYMPOSIUM OF THE WORLD ASSOCIATION OF VETERINARY LABORATORY DIAGNOSTICIANS (WAVLD)
Saskatoon - 17th June 2015
Recent breakthroughs in genome editing technology have led to a rapid adoption that parallels that seen with RNAi. And like RNAi, these methods are taking the scientific world by storm, with high profile publications in fields as diverse as HIV treatment, stem cell therapy, food crop modification and drug development to name but a few.
Critically, the endogenous modification of genes enables the study of their function in a physiological context. It also overcomes some of the artefacts that can result from established techniques such as transgenesis and RNAi, which have mislead researchers with false positives or negatives. Until recently however genome editing required considerable technical expertise, and consequently was a relatively niche pursuit.
In this talk we will look at how the latest developments in genome editing tools have changed this, with improvements in both ease-of-use and targeting efficiency, as well as a concomitant reduction in costs opening up these approaches to the wider scientific community.
Rapid adoption of the CRISPR/Cas9 system has for example led to a long list of organisms and tissues in which genetic changes have been made with high efficiency. Other technologies such as recombinant adeno-associated virus (rAAV) offer further precision, stimulating the cell’s high-fidelity DNA repair pathways to insert exogenous sequence with unrivalled specificity. Targeting efficiency can be improved still further by using the technologies in combination – genome cutting induced by CRISPR can significantly enhance homologous recombination mediated by rAAV.
Despite these rapid advances, some pitfalls remain, and so we’ll discuss some of the key considerations for avoiding these, ranging from simply picking the right tool for the job to designing an experiment that maximises chances of success.
Finally we’ll look at how genome editing is being applied to both basic and translational research, and in both a gene-specific and genome wide manner. For the study of disease associated genes and mutations scientists can now complement wide panels of tumour cells with genetically defined isogenic cell pairs identical in all but precise modifications in their gene of interest. The ease-of-design and efficiency of the CRISPR system is also being exploited for genome wide synthetic lethality screens, facilitating rapid drug target identification with significantly reduced risk of false negatives and off-target false positives. And again, further synergies are achieved when these approaches are combined to look for potential synthetic lethal targets in specific genomic contexts.
The CRISPR/Cas9 system has emerged as one of the leading tools for modifying genomes of organisms ranging from E. coli to humans. Additionally, the simple gene targeting mechanism of CRISPR technology has been modified and adapted to other applications that include gene regulation, detection of intercellular trafficking, and pathogen detection. With a wealth of methods for introducing Cas9 and gRNAs into cells, it can be challenging to decide where to start. In this presentation, Dr Adam Clore describes the CRISPR mechanism and some of the most prominent uses for CRISPR, along with methods where IDT technologies can assist scientists in designing, testing, and executing a variety of CRISPR-mediated experiments. For more informaton, visit: http://www.idtdna.com/crispr
Unraveling Excitation-Contraction Coupling: Simultaneous Measures of Intracel...InsideScientific
Join Patrick Schönleitner, PhD and Francisco Altamirano, PhD as they share their work involving simultaneous measurements of intracellular calcium, membrane voltage, and contractility of cardiomyocytes.
The heart’s electrical activity coordinates the contraction of the heart chambers to pump blood to other organs and sustain life. The movement of ions (charged atoms) through ion channels promotes voltage changes across the cardiomyocyte plasma membrane, known as action potentials. The arrival of an action potential depolarizes the plasma membrane and activates Ca2+ influx via L-type Ca2+ channels. Ca2+ then activates Ryanodine Receptors to release further Ca2+ into the cytosol and stimulate contraction. Both duration and shape of the cardiac action potential regulate Ca2+ fluxes and contractility in cardiomyocytes.
Cardiac excitation-contraction coupling is the process used to describe the progression from membrane depolarization to calcium influx and intracellular release to myocyte contraction. In the heart, these processes are tightly intertwined, and crucial information can be missed when recording them individually. Recent technological advances enable the simultaneous multi-parametric measurement of membrane voltage, intracellular calcium, and contractility to reliably capture the complex excitation-contraction cascade in great detail.
During the first half of this webinar, Patrick focuses on the theoretical background and address technical considerations and methodological limitations researchers need to consider to successfully run multi-parametric experiments and analyze the resulting data. Francisco then discusses how action potentials regulate Ca2+ fluxes to regulate cardiomyocyte contractile function. In addition, he discusses the advantages/limitations of mouse models and human inducible pluripotent stem cell (hiPSC)-derived cardiomyocytes to determine molecular mechanisms driving alterations in action potentials and Ca2+ handling.
The CRISPR/Cas9 system has emerged as one of the leading tools for modifying the genomes of organisms ranging from E. coli to humans. In this presentation, we discuss various methods for generating the crRNA and tracrRNA components that are required for guiding the Cas9 endonuclease to genomic targets. You will also learn how to optimize a new 2-part CRISPR RNA system from IDT that offers multiple benefits over other technologies.
Genome Editing Comes of Age; CRISPR, rAAV and the new landscape of molecular ...Candy Smellie
Information is no longer a bottleneck, emphasis is shifting to the ‘what does it all mean’
In a translational context we hope that by answering that question we will be able to is to characterise the genetics that drive disease, and indeed develop drugs and diagnostics that are personalised to patients.
Genome editing provides the link between the information here, and this outcome here, by allowing scientists to recapitulate specific genetic alterations in any gene in any living tissue to probe function, develop disease models and identify therapeutic strategies. So, not only do we now have unparalleled access to genetic information, but we now have the tools to most accuartely understand what this genetic information – with genome editing allowing us to explore the genetic drivers of disease in physiological models.
AAV is a single-stranded, linear DNA virus with a a 4.7 kb genome which for the purpose of genome editing is replaced almost in entirety with the targeting vector sequence (except for the iTRs)
It is in effect a highly effective DNA delivery mechanism
After entry of the vector into the cell, target-specific homologous DNA is believed to activate and recruit HR-dependent repair factors can induce HR at rates approximately 1,000 times greater than plasmid based double stranded DNA vectors, but the mechanism by which it achieves this is still largely unknown
By including a selection cassette can select for cells that have integrated the targeting vector, and then screen for clones which have undergone targeted insetion rather than random integration, which will generally be around 1%.
Presentation by Benedict Paten at GRC/GIAB ASHG 2017 workshop "Getting the most from the reference assembly and reference materials" on updates to the human reference assembly, GRCh38.
Course: Bioinformatics for Biomedical Research (2014).
Session: 2.1.1- Next Generation Sequencing. Technologies and Applications. Part I: NGS Introduction and Technology Overview.
Statistics and Bioinformatisc Unit (UEB) & High Technology Unit (UAT) from Vall d'Hebron Research Institute (www.vhir.org), Barcelona.
CRISPR/Cas9 gene editing is based on a microbial restriction system, that has been harnessed for genome targeting using only a short sequence of RNA as a guide.
The beauty of the system is that unlike protein binding based technologies such as Zinc Fingers and TALENs which require complex protein engineering, the design rules are very simple, and it is this fact that is allowing CRISPR to take genome engineering from a relatively niche persuit to the mainstream scientific community.
The principle of the system is that a short guide RNA, homologous to the target site recruits a nuclease – Cas9
This then cuts the dsDNA, triggering repair by either the low fidelity NHEJ pathway, or by HDR in the presence of an exogenous donor sequence.
High Efficiencies for both knockouts and knock-ins have been reported and whilst there are understandable concerns about specificity, new methodologies to address these are now being developed
The system itself is comprised of three key components
the Cas9 protein, which cuts/cleaves the DNA and
Two RNAs - a crispr RNA contains the sequence homologous to the target site and a trans-activating crisprRNA (or TracrRNA) which recruits the nuclease/crispr complex
For genome editing, the crisperRNA and TraceRNA are generally now constructed together into a single guideRNA or sgRNA
Genome editing is elicited through hybridization of the sgRNA with its matching genomic sequence, and the recruitment of the Cas9, which cleaves at the target site.
GRC Workshop at Churchill College on Sep 21, 2014. This is Michael Schatz's talk on the theory and practice of representing population data in graph structures.
Phylogenomic methods for comparative evolutionary biology - University Colleg...Joe Parker
Invited research seminar given to MSc students at University College Dublin on 24th October 2013.
I introduce the discipline of phylogenomics - comparative phylogenetic analyses of DNA sequences across genomes - and some of the applications and recent breakthroughs in the field.
As an in-depth case study I explain the methods and significance of our 2013 Nature paper on adaptive genotypic molecular convergence in echolocating mammals.
I then highlight some of the avenues of study on the frontiers of current research.
Accessing genetically tagged heterocycle libraries via a chemoresistant DNA s...Laura Berry
Presented at the Global Medicinal Chemistry and GPCR Summit. To find out more, visit:
www.global-engage.com
Andreas Brunschweiger, an Independent Group Leader at TU Dortmund, discusses the limitations of DNA-encoded compound libraries (DELs) and getting around these.
The CRISPR/Cas9 system has emerged as one of the leading tools for modifying genomes of organisms ranging from E. coli to humans. Additionally, the simple gene targeting mechanism of CRISPR technology has been modified and adapted to other applications that include gene regulation, detection of intercellular trafficking, and pathogen detection. With a wealth of methods for introducing Cas9 and gRNAs into cells, it can be challenging to decide where to start. In this presentation, Dr Adam Clore describes the CRISPR mechanism and some of the most prominent uses for CRISPR, along with methods where IDT technologies can assist scientists in designing, testing, and executing a variety of CRISPR-mediated experiments. For more informaton, visit: http://www.idtdna.com/crispr
Unraveling Excitation-Contraction Coupling: Simultaneous Measures of Intracel...InsideScientific
Join Patrick Schönleitner, PhD and Francisco Altamirano, PhD as they share their work involving simultaneous measurements of intracellular calcium, membrane voltage, and contractility of cardiomyocytes.
The heart’s electrical activity coordinates the contraction of the heart chambers to pump blood to other organs and sustain life. The movement of ions (charged atoms) through ion channels promotes voltage changes across the cardiomyocyte plasma membrane, known as action potentials. The arrival of an action potential depolarizes the plasma membrane and activates Ca2+ influx via L-type Ca2+ channels. Ca2+ then activates Ryanodine Receptors to release further Ca2+ into the cytosol and stimulate contraction. Both duration and shape of the cardiac action potential regulate Ca2+ fluxes and contractility in cardiomyocytes.
Cardiac excitation-contraction coupling is the process used to describe the progression from membrane depolarization to calcium influx and intracellular release to myocyte contraction. In the heart, these processes are tightly intertwined, and crucial information can be missed when recording them individually. Recent technological advances enable the simultaneous multi-parametric measurement of membrane voltage, intracellular calcium, and contractility to reliably capture the complex excitation-contraction cascade in great detail.
During the first half of this webinar, Patrick focuses on the theoretical background and address technical considerations and methodological limitations researchers need to consider to successfully run multi-parametric experiments and analyze the resulting data. Francisco then discusses how action potentials regulate Ca2+ fluxes to regulate cardiomyocyte contractile function. In addition, he discusses the advantages/limitations of mouse models and human inducible pluripotent stem cell (hiPSC)-derived cardiomyocytes to determine molecular mechanisms driving alterations in action potentials and Ca2+ handling.
The CRISPR/Cas9 system has emerged as one of the leading tools for modifying the genomes of organisms ranging from E. coli to humans. In this presentation, we discuss various methods for generating the crRNA and tracrRNA components that are required for guiding the Cas9 endonuclease to genomic targets. You will also learn how to optimize a new 2-part CRISPR RNA system from IDT that offers multiple benefits over other technologies.
Genome Editing Comes of Age; CRISPR, rAAV and the new landscape of molecular ...Candy Smellie
Information is no longer a bottleneck, emphasis is shifting to the ‘what does it all mean’
In a translational context we hope that by answering that question we will be able to is to characterise the genetics that drive disease, and indeed develop drugs and diagnostics that are personalised to patients.
Genome editing provides the link between the information here, and this outcome here, by allowing scientists to recapitulate specific genetic alterations in any gene in any living tissue to probe function, develop disease models and identify therapeutic strategies. So, not only do we now have unparalleled access to genetic information, but we now have the tools to most accuartely understand what this genetic information – with genome editing allowing us to explore the genetic drivers of disease in physiological models.
AAV is a single-stranded, linear DNA virus with a a 4.7 kb genome which for the purpose of genome editing is replaced almost in entirety with the targeting vector sequence (except for the iTRs)
It is in effect a highly effective DNA delivery mechanism
After entry of the vector into the cell, target-specific homologous DNA is believed to activate and recruit HR-dependent repair factors can induce HR at rates approximately 1,000 times greater than plasmid based double stranded DNA vectors, but the mechanism by which it achieves this is still largely unknown
By including a selection cassette can select for cells that have integrated the targeting vector, and then screen for clones which have undergone targeted insetion rather than random integration, which will generally be around 1%.
Presentation by Benedict Paten at GRC/GIAB ASHG 2017 workshop "Getting the most from the reference assembly and reference materials" on updates to the human reference assembly, GRCh38.
Course: Bioinformatics for Biomedical Research (2014).
Session: 2.1.1- Next Generation Sequencing. Technologies and Applications. Part I: NGS Introduction and Technology Overview.
Statistics and Bioinformatisc Unit (UEB) & High Technology Unit (UAT) from Vall d'Hebron Research Institute (www.vhir.org), Barcelona.
CRISPR/Cas9 gene editing is based on a microbial restriction system, that has been harnessed for genome targeting using only a short sequence of RNA as a guide.
The beauty of the system is that unlike protein binding based technologies such as Zinc Fingers and TALENs which require complex protein engineering, the design rules are very simple, and it is this fact that is allowing CRISPR to take genome engineering from a relatively niche persuit to the mainstream scientific community.
The principle of the system is that a short guide RNA, homologous to the target site recruits a nuclease – Cas9
This then cuts the dsDNA, triggering repair by either the low fidelity NHEJ pathway, or by HDR in the presence of an exogenous donor sequence.
High Efficiencies for both knockouts and knock-ins have been reported and whilst there are understandable concerns about specificity, new methodologies to address these are now being developed
The system itself is comprised of three key components
the Cas9 protein, which cuts/cleaves the DNA and
Two RNAs - a crispr RNA contains the sequence homologous to the target site and a trans-activating crisprRNA (or TracrRNA) which recruits the nuclease/crispr complex
For genome editing, the crisperRNA and TraceRNA are generally now constructed together into a single guideRNA or sgRNA
Genome editing is elicited through hybridization of the sgRNA with its matching genomic sequence, and the recruitment of the Cas9, which cleaves at the target site.
GRC Workshop at Churchill College on Sep 21, 2014. This is Michael Schatz's talk on the theory and practice of representing population data in graph structures.
Phylogenomic methods for comparative evolutionary biology - University Colleg...Joe Parker
Invited research seminar given to MSc students at University College Dublin on 24th October 2013.
I introduce the discipline of phylogenomics - comparative phylogenetic analyses of DNA sequences across genomes - and some of the applications and recent breakthroughs in the field.
As an in-depth case study I explain the methods and significance of our 2013 Nature paper on adaptive genotypic molecular convergence in echolocating mammals.
I then highlight some of the avenues of study on the frontiers of current research.
Accessing genetically tagged heterocycle libraries via a chemoresistant DNA s...Laura Berry
Presented at the Global Medicinal Chemistry and GPCR Summit. To find out more, visit:
www.global-engage.com
Andreas Brunschweiger, an Independent Group Leader at TU Dortmund, discusses the limitations of DNA-encoded compound libraries (DELs) and getting around these.
Avacta Life Sciences Affimers Presentation Global Protein Engineering Summit ...AvactaLifeSciences
Avacta Life Sciences Exhibits Affimers at Global Protein Engineering Summit
Avacta Life Sciences exhibited recently at the Global Protein Engineering Summit ("PEGS") where it presented its Affimer technology.
You can read more about Affimer technology here http://www.avactalifesciences.com
PEGS is considered to be the essential protein engineering meeting where commercial and academic progress in protein engineering is showcased and this year it attracted over 1800 delegates from across the globe to Boston. Avacta Life Sciences presented its Affimer technology for the first time at a PEGS meeting with technical exhibits and a presentation by the CSO, Paul Ko Ferrigno, entitled "Biological Recognition: Beyond the Antibody."*
The exhibition booth was busy with over 80 delegates talking to the Avacta Life Science management team over the four days of the summit. The feedback on the Affimer technology was very positive, in particular, the short development times and excellent stability were highlighted by delegates as key advantages of Affimers over antibodies. There was also a strong interest in Affimers from the management of companies developing biological therapeutics who were keen to learn more about the potential of Affimers as novel therapeutics.
In addition, several companies were interested in the use of Affimers as an alternative to antibodies in diagnostic devices, mainly because they could generate binders against new biomarkers much more quickly and evaluate them in higher numbers.
The benefits of Affimer microarrays for biomarker discovery also resonated with diagnostic developers who appreciated the advantage of being able to evaluate significantly larger numbers of potential biomarkers more cost and time effectively than by mass spectrometry. The potential of the arrays for multiplexed solutions for clinical diagnosis and monitoring during drug trials was also something that generated interest amongst those delegates.
Matt Johnson, Chief Technical Officer of Avacta Life Sciences commented: "It was great to experience face to face the level of interest in Affimers. The majority of people I spoke to were either having problems raising antibodies to their target of interest or just couldn't use antibodies because of the type of assays they wanted to perform. Many of the presentations focused around the use of antibody fragments for intra-cellular studies which is a rapidly growing area that holds great interest for drug and diagnostics developers. It is an area where there are clear advantages for Affimers over antibody fragments which don't behave well in the cytoplasm.
"The general enthusiasm around Affimers was very encouraging and the amount of interest generated by the potential of Affimers as therapeutics and by the Affimer arrays for biomarker discovery only reinforces my excitement around this new technology."
A recognition model of ACP-HCS interaction for programmed beta-branching in t...Rohit Farmer
Oral presentation at the Synthetic Biology of Antibiotic Production II (http://syntheticbio.esf.org), 30 August - 4 September 2014 at Sant Feliu de Guixols, Costa Brava, Spain.
RNA-Seq To Identify Novel Markers For Research on Neural Tissue DifferentiationThermo Fisher Scientific
Neural tissue differentiated and cultured from derived stem cells is expected to revolutionize the treatment of brain and spinal injuries and diseases. Critical for these cellular therapies is accurate control and monitoring of differentiation but current methods for such cell typing are limited to qPCR and immunocytochemisty (ICC) which is not sufficient to discriminate between the numerous (likely >100,000) possible neural cell-types. Research using RNA sequencing (RNA-Seq) permits the characterization and discovery of much-needed novel markers. To define the temporal transcriptional signature of neural stem cells, cultured human embryonic stem cells (H9) were compared to induced neural stem cells (NSCs) at d0, d7 and d14. Total RNA was isolated over the time course from the undifferentiated and differentiated cells. Ion Torrent™ libraries were created to profile expression of miRNAs and whole transcriptomes for each cell population. Multiplexed Ion Proton™ sequencing and Torrent Suite™ Software analysis yielded ≥2.5 million small RNA reads and ≥29 million whole transcriptome reads per sample. Cluster analysis of the RNA-Seq profiles indicates that the cell populations have characteristic molecular signatures. Among genes that are decreased in induced cells are OCT4 (POU5F1), JARID2, NANOG, consistent with the differentiation of iPSCs into neurons. Among genes that showed increased expressions are NTRK2, POU3F2, and a number of HOX family genes. Recently, Ion AmpliSeq™ Transcriptome Human Gene Expression Kit has been launched, and the results from this analysis corroborated with whole transcriptome RNA-Seq results.
1. Target Family-Centric Privileged Structures:
unexploited opportunities
for medicinal chemistry
Gerhard Müller
Senior Vice President
Medicinal Chemistry
Mercachem bv
gerhard.mueller@mercachem.com
2. • Bayer Pharma AG
• AstraZeneca AB
• H. Lundbeck A/S
• Janssen Pharma NV
• Merck KGaA
• Sanofi
• UCB Pharma SA
300.000 compound library
to be complemented with
200.000 novel compounds
400 novel scaffolds
non-mainstream
structural complexity
stereochemistry
scaffold diversity
challenging chemistry
€196 million pan-European drug discovery platform
WP9: Program Recruitment
(crowd-) sourcing of proposals from e.g. academia
WP10: Review & Selection
Library Selection Committee
(Prof. Adam Nelson)
WP11: Experimental Validation
Validation Management Team
(Mercachem)
WP12: Library Generation
5 SMEs involved, each SME:
8000 compounds annually based on
16 scaffolds annually
European Lead Factory – 200.000 novel „non-main stream“ compounds
attempts to surf the chemical space
2
as of May 1st:
680 scaffolds reviewed
> 390 scaffolds approved
~ 140 scaffolds validated
~50.000 compounds made
3. “What is clear is that certain “privileged structures“
are capable of providing useful ligands for more than
one receptor …“
“… judicious modification of such structures could be a viable
alternative in the search for new receptor agonists and
antagonists”
B. E. Evans et al. J. Med. Chem., 31, 2235 (1988)
A. A. Patchett et al. Annu. Rep. Med. Chem. 35, 289 (2000)
G. Müller, Drug Discovery Today 8, 681-691 (2003)
one ligand for more than one target system – very generic – no teaching!
navigating with privileged structures
N
N
N
H
O
S
F
tifluadom
k-opioid agonist
Nature, 298, 759 (1982)
CCK-A antagonist
Neurosci. Lett., 72, 211 (1982)
3
N
H
N
O
N
OH
N
N
O
Merck:
ORL1 antagonist
Roche:
ORL1 agonists
N
N
H
N
O
N N
NH
delete
bond
Dan Rich, 1990’s
Prevent hydrophobic collapse
5. J. Mol. Biol., 425, 662-677 (2013)
Convert GPCRs to soluble protein – biochemistry, biophysics, etc
G7 Therapeutics
5
Long –term apo-state stability:
No stabilizing ligand required
– no pre-empted activation state
Important for accessibility of fragments
Unbiased on functional signature
(ago, ant, inv.ago, etc)
Detergent solubility:
Isolated receptor protein in detergent micelles
Vapor-diffusion crystallization works out
Protein amenable to biochemistry and biophysics
SPR, NMR, TINS, FBLG, kinetics, thermodynamics, etc.
CHESS:
Cellular High-Throughput Encapsulation Solubilising and Screening
Class A GPCRs
Neurotensin 1 receptor (NTS1)
κ-opioid (KOR)
Tachykinin receptor 1 (NK1)
Oxytocin
Adrenergic receptors
α1A
α1B
Class B GPCR
Parathyroid hormone receptor 1
(PTH1)
Fig. S2. Quality of the σA-weighted 2FO-FCelectron density map contoured at 1.2 σ. Stere
Directed evolution – error-prone PCR
6. O
N
O
O
Annelated ring systems – novelty analysis ongoing
Fsp3-rich carbon skeletons
annelation-constrained macrocycles
• sp3-rich
• stereochemical complexity
• 3D shape
• non-main stream
6
annelated
3796 compounds
395 BM scaffolds
401 unique targets
[5:5] cyclopentan-pyrolidine [6:5] piperidino-pyrrolidine
[5:5] pyrrolidino-diazolidine-dione g-exo-homo Proline
[6:7] reverse turn mimic
[6:5] reverse turn mimic
7. P. Furet et al. J. Comp.-Aid. Mol Des. 1995, 9, 465-472
P. Traxler, Exp.Opin. Ther. Patents 1998, 8, 1599-1625
N
NN
H
N
H
O
N
N
N
stabilizing inactive kinase conformation
NH
N
NH
O
O
H
O
deep
pocket
Gleevec : c-Abl
type II
NH
N
NH
O
O
H
O
back pocket
N
Cl
Cl
O
N
NN
H
S
competitive inhibition of active state
PD173955 : c-Abl
1m52.pdb
PD173955 : c-Abl
1iep.pdb
Gleevec : c-Abl
DFG-in (I) vs. DFG-out (II)
activation loop undergoes significant structural rearrangement
type I
DFG-in
DFG-out
7
8. DFGin
DFGout
many „DFG-in-between“ ?
misleading terminology
A.P. Kornev, N. M. Haste, S. S. Taylor, L. F. Ten Eyck, Proc. Natl. Acad. Sci., 103, 17783 (2006)
A.P. Kornev, S. S. Taylor, L. F. Ten Eyck, Proc. Natl. Acad. Sci., 105, 14377 (2008)
A.P. Kornev, S. S. Taylor, Biochim. Biophys. Acta, 1804, 440-444 (2010)
H.S. Meharena, et al, PLOS Biology, 11 (10), 1-11 (2013) 8
9. hydrophobic spine
comparison of intact and disrupted spines
O
N
N
H
O
N
H
N
H
O
Cl
CF3
intact R-spine disrupted R-spine (CDK8)
9
active kinase
type I inhibitors
fast kinetics
inactive kinase
non-type I inhibitors
slow kinetics (t½ 8 h)
10. Retro-Design Approach: B2F (back-to-front)
Sets out with scaffolds rather than leads
Disrupt hydrophobic spine
Long residence time on target; slow koff
Option for exploration of novel IP space
Option for increased selectivity
NH
N
NH
O
O
H
O
“Retro Design“ approach
targeting conformational states by deep pocket-directed scaffolds
10
11. UPR as one major cause for neurodegeneration
AD disease mechanisms: UPR
ER
UPR (unfolded protein response):
• cellular stress pathway
• protein misfolding in ER = stress
• stress sensors:
• PERK, ATF6, IRE1
• „sense“ mis-folded proteins
• expression of Chaperons – assist in protein folding
11
pPERK
Phospho-Tau (AT8)
Hippocampus anatomy
PERK
12. N
N N
NH2
N
O
N
CH3
F
GSK2656157 (IC50: 0.8 nM)
development candidate
WO2011119663A1
J.Med.Chem. 2012, 55, 7193-7207
Med.Chem.Lett. 2013, 4 (10), pp 964–968
IC50: 2.72 nM
Novel, IP-free scaffold
12
4x7n4g313qd2
R-Sp2 and R-Sp3 inhibition mode = f(inhibitor)
hydrophobic spine topology
13. CDK8 in oncology
marked modulation of selectivity profile within deep pocket - controversial
Steady increase in selectivity throughout the consecutive compound generations
Initial generation Novel generation Latest generation
13
See poster 25
14. 14
IC50(M)
similar potency;
huge difference in residence time
similar residence time;
difference in potency
Residence time (h)
similar potency;
huge difference in residence time
Escape Trajectory: retrograde induced-fit mechanims of dissociation
IC50 / koff Correlation (?)
With appreciation to R. Buijsman, NTRC, Oss, NL
Ponatinib
d
on
off
K
k
k
P + L
PL
ΔG‡
on
ΔGd
ΔG‡
off
RT/G-
on
‡
on
ek D
RT/G-
off
‡
off
ek D
/RTG-Δ
d eK d
Bindingcoordinate
15. 15
compound synthesis in DNA-cavity
Holliday Junction
50 Mio compound library
linear and branched compounds
spiked with PPI motifs
• turns (beta and gamma)
• strands (exteded conformations)
• Trp, Arg, Tyr
Protein-Protein Interaction-targeted Library
DNA-encoded Library Technology
16. p38a as a prototypic kinase amenable to type II inhibition
Tri-peptide mimetics for kinases ?
16
IC50>2µM
% remaining activity @ 2µM compound concentration
0
25
50
75
100
19. rapid interrogation of SAR by point mutations in compound structure
Advantage of modular chemistry
N
H
O
N
N
NH2
O
N
H
NH2
O
required ?
R-groups
heterocycles
ring size
acceptor !
ring size
heterocycles
R-groups
required ?
NH-Me, Cl, F
Me, CF3
stabilize kinked cofo!
stereochemistry
small substituents
spacing
heterocycles
R-groups
ring size
scan pocket better
change physchem
stabilize cofo
donor required !
spacing, b-AA
NH-R, explore pocket
Weeks 1 2 3 4 5 6
step 1: amide coupling
step 1: purification
Step 2: Pg removal
Step 2: purification
Step 3: amide coupling
Step 4: purification
Unfold near-to-complete SAR in 6 weeks
of chemistry
Iterative analogue libraries
19
20. summary
• pharmaceutically relevant targets cluster into densely
populated families
• high Fsp3 compounds pursuing an indirect approach
chemical similarity correlates with biological similarity
• target family-centric rationales allow for imprinting
family-wide commonalities into new scaffolds
• functional attributes can be pre-engineered
you might want to look for slow-off compounds
• despite the run on epigenetic targets, the days of kinases
are not yet over
• DNA-encoded library approaches uncover truly novel
chemotypes for target families
20
consider binding kinetics
optimize residence time for multiple reasons
Bob Copeland (CSO Epizyme Inc.)
10th Swiss med chem course, Leysin, 2012
„any chemist reporting IC50‘s should
be boiled in oil!“
21. www.mercachem.com
Arjen Brussard
Tim Moser
Christoph Schächtele
Michael Kubbutat
Nils Jakob Vesten Hansen
Tara Heitner Hansen
Kiyoshi Takayama
Tomoko Shimizu
Jürgen Bajorath
Dagmar Stumpfe
Carlo Bertozzi
Lutz Kummer