EPIGENETICS (i) Cell Biology

(ii) New Platform in Drug Discovery

Dr. Ana M Roa

Plasma membrane

Nucleus

Cytoplasm

Cell: functional units of all living organisms

Cell Membrane

Cytoplasm

CytoskeletonKeep cellular structureCellular divisionMigration & Adhesion

Subcelular OrganulesMultiple Functions

Subcellular organulesMultiple functions

(inflammation, cancer)

(metabolic disorders,cancer)

Chomosomes: DNA & Proteins

NucleusGene Expression

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.“

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

Epigenetics and Environment

Certain environmental and dietary factors have been linked to abnormal changes in epigenetic pathways in experimental and epidemiological studies.

Food rich on methyl donors

Normal diet

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.

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.

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.

Epigenetics in Plants

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

Chromodomains Chromodomains ReadersReaders

ReadersReadersBromodomainsBromodomains

HDACsHDACsSirtuinsSirtuins

Epigenetic alterations associated with disease

Vol 28 Nº10 OCTOBER 2010 nature biotechnology

Epigenetic alterations associated with disease

Vol 28 Nº10 OCTOBER 2010 nature biotechnology

Epigenetic Inhibitors for Therapies

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

Chemical structures of selected compounds that target epigenetic modifications

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

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

JmjD family HTS steps

1. Assay development of a Biochemical Assay2. Testing of pharmacology3. Testing the Robustness of the assay in the presence of

compounds4. Determination of the Quality Control parameters and the

statistical expected for the primary screening campaign5. If all OK: Launch the HTS campaign (2M compounds)6. Data analysis with specific software7. Hits selection using chemo-physical properties 8. Hits confirmation9. Determination of the potency and selectivity of the

confirmed actives10. Progress the best compounds to the Critical Path

designed to determine real potential drugs

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

BRDs control gene transcription

Challenges with targeting epigenetic readers

• BET Bromodomain Inhibition as a Therapeutic Strategy to Target c-MycCell 01 September 2011 • RNAi screen identifies Brd4 as a therapeutic target in acute myeloid leukemiaNature (2011) 1034• Selective inhibition of BET bromodomainsNature 468, 1067–1073 (23 Dec 2010)

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

Many thanks for your attention !