Una revisión de los conocimientos fundamentales de la biología de la célula. La epigenética
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Conferencia de la Dra. Ana María Roa, Bióloga Molecular, sobre Epigenética, impartida en la Universidad Popular Carmen de Michelena de Tres Cantos el 1 de marzo de 2013. Más información en: http://www.universidadpopularc3c.es/index.php/actividades/conferencias/event/448-conferencia-una-revision-de-los-conocimientos-fundamentales-de-la-biologia-de-la-celula-la-epigenetica
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Una revisión de los conocimientos fundamentales de la biología de la célula. La epigenética
- 1. EPIGENETICS (i) Cell Biology (ii) New Platform in Drug Discovery Dr. Ana M Roa
- 2. Plasma membrane Nucleus Cytoplasm Cell: functional units of all living organisms
- 4. Cytoplasm Cytoskeleton Keep cellular structure Cellular division Migration & Adhesion Subcelular Organules Multiple Functions Subcellular organules Multiple functions (inflammation, cancer) (metabolic disorders, cancer)
- 5. Chomosomes: DNA & Proteins Nucleus Gene Expression
- 6. What is Epigenetics? Epigenetics is the study of heritable changes in gene expression or cellular phenotype, caused by mechanisms other than changes in the underlying DNA sequence – epi- (Greek: επί- over, above, outer) -genetics. It refers to functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence. – DNA methylation and histone modification, both of which serve to regulate gene expression without altering the underlying DNA sequence. – These changes may remain through cell divisions for the remainder of the cell's life and may also last for multiple generations. “The study of the mechanisms of temporal and spatial control of gene activity during the development of complex organisms.“
- 7. Epigenetics: The life cycle of the epigenome A number of mechanisms controlling chromatin remodelling include DNA methylation & histone modification Dysregulation of any of them is implicated in human disease
- 9. Epigenetics and Environment Certain environmental and dietary factors have been linked to abnormal changes in epigenetic pathways in experimental and epidemiological studies.
- 10. Food rich on methyl donors Normal diet
- 12. Epigenetics in Microorganisms • The filamentous fungus Neurospora crassa is a model system for understanding the control and function of DNA methylation. In this organisms, DNA methylation is associated with a genome defense system called RIP (repeat-induced point mutation) and silences gene expression by inhibiting transcription elongation.[75] • Bacteria make use of DNA adenine methylation as an epigenetic signal. DNA adenine methylation is important in bacteria virulence in organisms such as Escherichia coli, Salmonella, Vibrio, Yersinia, Haemophilus, and Brucella.
- 13. Epigenetics in Tuberculosis 2 billion people worldwide are infected with tuberculosis, 10 million of whom fall ill each year. Tuberculosis is the leading cause of death among people co- infected with HIV, the virus that causes AIDS, leading to some half-million deaths annually among those co-infected. Genetic and Epigenetic Variation in Mycobacterium tuberculosis determine whether M. tuberculosis varies, genetically and/or epigenetically, during the course of single infections and whether this variation is subject to selection by the host immune response. Hypermutability and the Acquisition of Multidrug Resistance account for the emergence of extended drug resistance in some clinical strains.. Current studies on Functional genomics of M.tuberculosis.
- 14. Epigenetics in Malaria Malaria is a major public health problem in many developing countries, with the malignant parasite Plasmodium falciparum causing the most malaria- associated mortality. The chromatin-mediated mechanisms underlie many cellular processes in the parasite's development The distinction of the parasite's chromatin modification machinery from those of its mammalian hosts makes it a promising target for antimalarial chemotherapy. HDACs have been explored as potential candidates for antimalarials. With the identification of most of the PTMs, modifiers, and “readers,” future studies will allow to advance toward a mechanistic understanding of chromatin-mediated gene regulation in the malaria parasite. Elucidation of the epigenetic pathways in malaria parasites not only will help us to understand gene regulation in this unicellular parasite but also will provide insights into many parasite-specific phenomena such as antigenic variation and alternative invasion pathways, which may ultimately lead to the development of novel control measures targeting host- parasite interactions.
- 16. Epigenetic Targets: Histone modification Histones KDMsKDMs Chromosome 1 in 3-years-old identical twins (left) and in 50- years-old (right) P.N.A.S. Divergence with time Writers, Erasers and Readers in the Book of Life ChromodomainsChromodomains ReadersReaders ReadersReaders BromodomainsBromodomains HDACsHDACs SirtuinsSirtuins
- 17. Epigenetic alterations associated with disease Vol 28 Nº10 OCTOBER 2010 nature biotechnology
- 18. Epigenetic alterations associated with disease Vol 28 Nº10 OCTOBER 2010 nature biotechnology
- 19. Epigenetic Inhibitors for Therapies
- 20. Therapeutic areas targeting epigenetic enzymes Therapeutic area % R&D Oncology 71 Metabolic disease/Diabetes 38 CNS/Neurodegeneration/Pain 29 Inflammatory disease/Autoimmune 29 Cardiovascular 28 Anti-infectives/Anti-viral 16 Others… 15
- 21. Chemical structures of selected compounds that target epigenetic modifications
- 22. Looking for new active molecules in Drug Discovery Developing robust and uHTS assays for searching small- molecule inhibitors targeting key epigenetic proteins is crucial in the discovery process of clinically relevant compounds
- 23. Epi-Enzymes Jumonji family (JmjD) of KDMs Able to demethylate 3, 2 &1 methyl groups Largest family of HDMs Uniquely require α-ketoglutarate and Fe(II) At least 30 JmjDs in the human genome with unique target specificities. Selective inhibition of JmjD may be sufficient for modulation of some diseases JmjD1a JmjD1b JmjD2a JmjD2b JmjD2c JmjD2d - JmjD3 - Gene Repression Human H3 N-ARTKQTARKSTGGKAPRKQLAKAARKSAPATGGVKKPHR….4 9 27 Background
- 24. JmjD family HTS steps 1. Assay development of a Biochemical Assay 2. Testing of pharmacology 3. Testing the Robustness of the assay in the presence of compounds 4. Determination of the Quality Control parameters and the statistical expected for the primary screening campaign 5. If all OK: Launch the HTS campaign (2M compounds) 6. Data analysis with specific software 7. Hits selection using chemo-physical properties 8. Hits confirmation 9. Determination of the potency and selectivity of the confirmed actives 10. Progress the best compounds to the Critical Path designed to determine real potential drugs
- 25. Bromodomains recognise acetylated lysine residues within histone tails 45 human bromodomains Targets proteins identified as BET family: – Brd-2, -3, -4 Epi-Readers BET family bromodomains Bromodomains Background Diversity in loop region (selectivity) High species homology at AcK JBC,2007
- 26. BRDs control gene transcription
- 27. Challenges with targeting epigenetic readers
- 28. • BET Bromodomain Inhibition as a Therapeutic Strategy to Target c-Myc Cell 01 September 2011 • RNAi screen identifies Brd4 as a therapeutic target in acute myeloid leukemia Nature (2011) 1034 • Selective inhibition of BET bromodomains Nature 468, 1067–1073 (23 Dec 2010)
- 29. Future trends….. Nuclear magnetic resonance (NMR) Isothermal Titration Calorimetry (ITC) Dynamic light scattering Surface plasmon resonance (SPR) Dual polarisation interferometry Microscale thermophoresis (MST) To assess whether (i) the compound binds effectively to the target, (ii) the stoïchiometry of binding, (iii) any associated conformational change and (iv) to identify promiscuous inhibitors. New Technologies for Biophysical testing
- 30. Many thanks for your attention !
Editor's Notes
- The emerging field of epigenetics, a new field of science that describes how the selective regulation of chromatin state controls key cellular activities such as gene expression, cell fate decisions, and DNA damage repair. Miscues in these normal events contribute to the development of human diseases such as cancer, inflammation, autoimmune diseases and neurodegenerative diseases. Drugs that reverse these miscues should provide high-impact medical benefit. Constellation Pharmaceuticals is building the premier chromatin therapeutics company through the discovery and development of drugs targeting epigenetic function and associated disease biology.
- The cytoplasm of a cell is surrounded by a cell membrane or plasma membrane . The plasma membrane in plants and prokaryotes is usually covered by a cell wall. This membrane serves to separate and protect a cell from its surrounding environment and is made mostly from a double layer of lipids (hydrophobic fat-like molecules) and hydrophilic phosphorus molecules. Hence, the layer is called a phospholipid bilayer, or sometimes a fluid mosaic membrane. Embedded within this membrane is a variety of protein molecules that act as channels and pumps that move different molecules into and out of the cell. The membrane is said to be 'semi-permeable', in that it can either let a substance (molecule or ion) pass through freely, pass through to a limited extent or not pass through at all. Cell surface membranes also contain receptor proteins that allow cells to detect external signaling molecules such as hormones.
- El citoplama contiene estructuras subcelulares y líquido citoplasmático Citoesqueleto: Mantiene la estructura celular division migración y adhesion Mitocondrias Retículo endoplásmic, etc Liquido citoplasmático Kinasa, fostatasas,….
- El nucleo: transmision de información genética durante la división celular Procesos de division celular anómalos pueden dan lugar a crecimientos celulares “descontrolados” (tumores) Cromosomas: ADN y proteinas. Código genético y sitios de unión específicos para factores reguladores de replicación y transcripción
- The DNA strand in each cell is wound around a protein complex called the histone to compact the DNA into the cell nucleus (chromatin) . Changes in chromatin structure due to either chemical modification of the DNA (methylation) or changes in DNA-histone interactions help control gene expression, silencing or activating genes. Improper gene expression leads to the improper production or non-production of proteins which are the source of most diseases and disorders. Various chemical modifications to the histones cause the DNA to wrap more tightly or loosely around the histones. These chemical modifications are directly responsible for making the DNA accessible or inaccessible to control gene regulation “histone post translational modifications. The post translational modifications which usually take place on the “tails“of histones include methylation, acetylation, phosphorylation and ubiquitination. An increasing number of these newly identified enzymes have been associated with neurodegenerative disorders, metabolic diseases, inflammation, and most notably, cancer. “ The difference between genetics and epigenetics can probably be compared to the difference between writing and reading a book. Once a book is written, the text (the genes or DNA) will be the same in all the copies. However, each individual reader may interpret the story slightly differently. Conrad Waddington (1905-1975) is often credited with coining the term epigenetics in 1942 as “the branch of biology which studies the causal interactions between genes and their products, which bring the phenotype into being”. Epigenetics appears in the literature as far back as the mid 19th century, although the conceptual origins date back to Aristotle (384-322 BC). He believed in epigenesis: the development of individual organic form from the unformed. This controversial view was the main argument against our having developed from miniscule fully-formed bodies. Even today the extent to which we are preprogrammed versus environmentally shaped awaits universal consensus. The field of epigenetics has emerged to bridge the gap between nature and nurture. In the 21st century you will most commonly find epigenetics defined as ‘the study of heritable changes in genome function that occur without a change in DNA sequence‘. But what do the scientists that work in this rapidly expanding research field have to say?
- The study of the epigenetic mechanism in malaria parasites is still in its infancy, and most knowledge gathered thus far is from focused studies of antigenic variation in P. falciparum. The available information clearly indicates that chromatin-mediated mechanisms underlie many cellular processes in the parasite's development. From an evolutionary point of view, the malaria parasites have a typical nucleosome organization, numerous histone PTMs, and a large catalogue of conserved chromatin modification and remodeling machineries and histone-binding modules, suggesting conserved epigenetic mechanisms in these early-branching protozoan parasites. Therefore, in-depth studies of the epigenome of the malaria parasites will contribute to a better comprehension of how chromatin-mediated mechanisms have evolved. In addition, the malaria parasite also possesses distinct histone PTMs and divergent histone variants, and their combination is expected to generate a complex yet different “histone code.” Besides, among the “writers” and “readers” of the “histone code” in this parasite, many are unique or contain unique domains, but only a countable few have been characterized so far. Thus, elucidation of the epigenetic pathways in malaria parasites not only will help us to understand gene regulation in this unicellular parasite but also will provide insights into many parasite-specific phenomena such as antigenic variation and alternative invasion pathways, which may ultimately lead to the development of novel control measures targeting host-parasite interactions. More importantly, the distinction of the parasite's chromatin modification machinery from those of its mammalian hosts makes it a promising target for antimalarial chemotherapy. For example, HDACs have been explored as potential candidates for antimalarials, and many HDAC inhibitors have potent antimalarial activity (1). With the identification of most of the PTMs, modifiers, and “readers,” future studies will need to characterize and integrate these components and to advance toward a mechanistic understanding of chromatin-mediated gene regulation in the malaria parasite.
- Epigenetics: the bookmark in the book of life Epigenetics: inheritable changes on gene expression, and hence the proteome and the phenotype, that are not based upon changes in the DNA sequence Redundacy and divergence in enzymes; selectivity: substrate, differential expression Epigenetics allow different interpretations of the fixed genetic code and result in different read-outs, dependent upon the variable conditions” Thomas Jenuwein (Vienna,Austria)
- In the picture below, certain key functions are selected of the lead identification and lead optimization process. Clinical and development phases are neglected because MDL does not provide solutions in these areas. The pre-clinical area is important for the near future, because here cheminformatics, genomics, combinatorial chemistry and high throughput screening can make drastic improvements. Such improvements are needed to propel the industry to an seemingly impossible three to eightfold increase in output of new drugs. ( ...some companies are heading for a crash unless they can rethink their approach so completely that R&D costs and lead times plummet, generate additional sales revenues with blockbuster drugs or move into brand new markets.... there could be as few as 13 top companies by the year 2005. Sir Mark Richmond, Glaxo's former Head of Research Worldwide, goes further. "We could see just three...." taken from "Pharma 2005, An Industrial Revolution in R&D from PriceWaterhouseCoopers.) The workflow of the development of a new pharmaceutical entity can be pictured by a huge funnel, which allows the easy input of information that is then compressed to be used to produce a new drug. MDL concentrates on the pre-clinical phase. There you will deal with many chemical structures and any workflow and information system must be based on handling chemical structures. Handling the chemical structure, also the chemical reaction, was pioneered by MDL
- Cartoon of the reaction and biological activity, and the assay principle
- Approx. 110 residue domain; 4 helices, 2 key loops Bromodomain External Terminal