Frontiers in epigenetic chemical biology

09:05-09:30 From antibiotics to histone deacetylase inhibitors: elucidating and exploiting biosynthetic protein-protein interactions

Professor Gregory Challis, University of Warwich, UK; Monash University, Australia

Abstract

Modular polyketide synthases (PKSs), nonribosomal peptide synthetases (NRPSs) and hybrid PKS-NRPSs are remarkable molecular machines that are responsible for the assembly of numerous bioactive natural products with a range of important applications in medicine and agriculture. These assembly line multienzymes typically consist of several subunits, which must interact specifically with each other to ensure a high degree of fidelity in the overall biosynthetic process. Over the past decade it has become apparent that interactions between subunits are typically mediated by structurally complementary N- and C-terminal docking domains that engage in specific protein-protein interactions.

In this lecture, Professor Challis will describe the efforts to understand the role played by specific protein-protein interactions in a hybrid PKS-NRPS responsible for the assembly of enacyloxin IIa, an unusual antibiotic with promising activity against Acinetobacter baumannii. The results of these studies reveal that a pair of complementary docking domains, previously thought to mediate subunit interactions in only a handful of NRPSs and hybrid PKS-NRPSs, are in fact present in many different systems, including the assembly line responsible for the biosynthesis of the clinically-used histone deacetylase inhibitor romidepsin. It has been shown that non-cognate docking domains belonging to this class are able to interact productively with each other. The implications of these findings for biosynthetic engineering approaches to the production of novel analogues of romidepsin and other clinically-important natural products will be discussed.

09:30-09:45 Discussion

09:45-10:15 HDAC inhibitors, progress and prospects

Professor A. Ganesan, University of East Anglia, UK

Abstract

Acylation of lysine residues is perhaps the most important post-translational modification of proteins after phosphorylation. In nucleosomes, the N-terminal tails of histone proteins primarily undergo acetylation but also linkage to a variety of short chain carboxylic acids as well as small proteins such as ubiquitin and SUMO. The action of lysine deacylases, grouped into histone deacetylases (HDACs, 11 human isoforms) and sirtuins (SIRTs, 7 human isoforms) reverses protein acylation and promotes transcriptional gene silencing. As disruption of the balance between acylation and deacylation is manifested in many human diseases, HDAC inhibition has become an important drug discovery strategy. Five inhibitors that reversibly bind to the active site zinc cation are approved drugs for cancer and multiple other candidates are in clinical trials. The presentation will focus on HDAC inhibitors from the group based on natural products and synthetic scaffolds with examples of isoform selectivity and therapeutic indications outside cancer.

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10:15-10:30 Discussion

10:30-11:00 Coffee

11:00-11:30 Metabolic regulation of SIRT2, a dual-specificity deacylase

Professor Minoru Yoshida, RIKEN, Japan

Abstract

Acetylation of proteins represents a widespread phenomenon from histones to many other proteins and is involved in fundamental processes such as epigenetics and signal transduction. SIRT2 is a member of the NAD-dependent lysine deacetylase family and regulates several biological processes such as epigenetics and cell migration. The activity of SIRT2 is upregulated by the cellular NAD+, but the existence of endogenous metabolites inhibiting SIRT2 remains unknown. The group recently identified selective SIRT2 inhibitors from the RIKEN NPDepo chemical library. X-ray crystal structure of SIRT2 in complex with the synthetic inhibitors revealed that SIRT2 possesses a deep hydrophobic cavity behind its substrate-binding pocket, to which a long-chain fatty acyl group may potentially bind. Indeed, the crystallographic analysis of SIRT2 in complex with myristoylated lysine peptide revealed that the myristoyl group of the peptide was accommodated in the hydrophobic cavity similar to that created by the SIRT2 inhibitors. Furthermore, the group could detect the reaction intermediate in the co-crystal in the presence of NAD+, demonstrating the catalytic mechanism for long chain de-fatty acylation by SIRT2. These observations prompted them to screen cellular metabolites for endogenous SIRT2 inhibitors. In this presentation, Professor Yoshida will report identification of lipid metabolites that strongly inhibit SIRT2 and their mode of action revealed by X-ray crystallography. The observation suggests that dual-specificity deacylase activity of SIRT2 is regulated by a variety of endogenous metabolites.

11:30-11:45 Discussion

11:45-12:15 Regulation of protein biosynthesis by oxygen

Professor Christopher Schofield FRS, University of Oxford, UK

Abstract

Oxygenases are involved in the biosynthesis of a range of natural products where they often catalyse synthetically impossible reactions such as occur in beta-lactam biosynthesis. Oxygenases also play important roles in the physiology of humans. In animals the response to limiting oxygen availability is mediated by the hypoxia inducible transcription factor (HIF). Both the levels and activity of HIF are regulated by its post-translational hydroxylation of conserved prolyl and asparaginyl residues. These modifications are catalysed by Fe(II) and 2-oxoglutarate dependent oxygenases which are also involved in nucleosome and ribosome modifications. The talk will discuss the biochemical and structural features that enable 2OG oxygenase catalsyis in a range of biosynthetic/biological systems, and to act as sensors in some contexts. The talk will describe evidence that post-translational hydroxylations, are widespread.

12:15-12:30 Discussion

13:30-14:00 Design and activity of DNA methylation inhibitors in cancer cells

Dr Paola B. Arimondo, CNRS ETaC, France

Abstract

Dr Arimondo’s group has designed novel non-nucleoside inhibitors of DNA methyltransferases which are able to demethylate and reactivate tumour suppressor genes in cancer cell lines. The group applied three chemical strategies: high-throughput screening of chemical libraries; rational drug design based on molecular modelling; and the pharmocomodulation of known inhibitors. The cellular consequences of this DNA demethylation are studied in comparison to nucleoside inhibitor decitabine. Altogether, four new families of DNMT inhibitors were optimised and studied. These studies provide insights of the next-generation of DNMT inhibitors.

14:00-14:15 Discussion

14:15-14:45 Identification and characterisation of methylated deoxyadenosines in vertebrates

Dr Magdalena Koziol, University of Cambridge, UK

Abstract

Methylation of cytosine deoxynucleotides is a well-established epigenetic mark, but in higher eukaryotes much less is known about modifications affecting other deoxynucleotides. Dr Koziol reports the detection of N-6-methyl-deoxyadenosine in vertebrate DNA, such as frogs, but also in other species including mouse and human. This methylome analysis reveals that dA^6m is widely distributed across the eukaryotic genome, is present in different cell types, but commonly depleted from transcriptional start sites, in particular gene exons. Dr Koziol discusses an association between dA^6m and genes involved in neuronal pathways, as well as identify a potential dA^6m motif in the vertebrate genome. Overall, Dr Koziol believes dA^6m could be implicated in regulating gene transcription, but more work is required to elucidate this function in vertebrates.

14:45-15:00 Discussion

15:00-15:30 Tea

15:30-16:00 Deregulation of non coding RNA in cancer and their interactions with histone modifiers

Dr María Berdasco Menéndez, Bellvitge Institute for Biomedical Research, Spain

Abstract

Protein-coding genes are the best studied sequences of the genome; however, coding exons only account for less than a 1.5% of the genome. A large fraction of the genome is constituted by non-coding elements that might have critical functional roles, including gene regulatory regions, origins of replication or non-coding RNAs (ncRNAs), among others. Although initially considered non-functional elements (‘the dark matter’), recent evidence suggests that ncRNAs play major biological roles in cellular biology, development and homeostasis. Several studies have demonstrated that lncRNAs are deregulated in cancer tissues and other human disorders. In fact, ncRNAs are considered fundamental epigenetic players, together with CpG methylation and histone modifications, as differential expression of ncRNA might play a role in targeting epigenetic writers and erasers to their target genes. Furthermore, ncRNAs may themselves be targets of epigenetic disruption. In this regard, it is well established that several ncRNAs (including long-ncRNAs and microRNAs) are silenced by CpG hypermethylation of their regulatory regions in cancer. As a consequence of their increasing role in human pathogenesis, new uses of ncRNAs biology are being explored including their potential as biomarkers of prognosis and response to chemotherapy but also as therapeutic targets.

16:00-16:15 Discussion

16:15-16:45 DNA bases beyond Watson and Crick

Professor Thomas Carell, Ludwig Maximilians Universität München, Germany

Abstract

Our genetic system is constructed based on four canonical nucleobases: adenosine, cytosine, guanosine and thymine. Next to these bases a number of non-canonical bases have recently been discovered that seem to regulate transcriptional activity. This information level is established by the new DNA bases hmC, fC and caC. In the lecture Professor Carell will discuss the most recent results regarding the distribution and function of these new bases. He will show that the lack of oxygen, as present in many tumour tissues, is strongly limiting formation of these bases and that this in turn effects the activity of key oncogenes in oxygen derived tumours. Professor Carell will show data that link formation of the new bases to other metabolic processes and he will show isotope tracing studies that reveal the faith of the new bases in the genome after their formation. Many of the results rely on the use of mass spectrometry and this will be explained together with the chemical synthesis of phosphoramidites of the new bases and the development of new reagents for mass spectrometric based proteomics studies.

16:45-17:00 Discussion

09:00-09:30 Epigenetic approaches to overcome cancer drug resistance: a moving target

Professor Robert Brown, Imperial College London, UK

Abstract

Epigenetic events, somatically inherited through cell division, are potential drivers of acquired drug resistance in cancer. The high rate of epigenetic change in tumours generates diversity in gene expression patterns that can rapidly evolve through drug selection during treatment, leading to acquired resistance. Furthermore, persistent cell populations may survive drug exposure and have changes in gene expression associated with histone marks and chromatin modifying enzymes: these cells are reversibly drug tolerant, but can acquire resistance after further growth that becomes fixed by DNA methylation. Pharmacological reversion of repressive DNA and histone epigenetic marks can lead to re-sensitisation of tumours to chemotherapy in experimental models and some epigenetic drugs have entered clinical trials as resistance modulators with mixed success. Given the complexity and diversity of tumours, it is perhaps unrealistic to expect that drug resistance can ever be completely overcome. However, prevention of emergence of resistance by targeting epigenetically poised states, rather than reversal of a fixed epigenetic state, may be a more fruitful approach. Patient-derived xenograft models of acquired resistance, combined with gene targeted approaches such as CRISPR RNA-guided endonuclease Cas9, locus specific editing of histone modifications or chemical biology approaches using an epigenetic targeted pharmacopeia, that allow functional consequences of gene or mechanism specific intervention to be examined, has potential to provide innovation and insight into mechanisms of acquired resistance.

09:30-09:45 Discussion

09:45-10:15 Rewriting a gene's epigenetic signal at will

Professor Marianne Rots, University Medical Center Groningen, The Netherlands

Abstract

Epigenome-wide Association Studies have resulted in epigenetic mutations which might serve as diagnostic, prognostic, or even therapeutic targets. To functionally validate and further exploit such epigenetic biomarkers as therapeutic targets, epigenetic editing provides a powerful approach.

In epigenetic editing, epigenetic writers or erasers are fused to DNA binding platforms, such as engineered Zinc Finger Proteins (ZFPs) or the recently introduced CRISPR-Cas platform. CRISPR-Cas is based on designed RNA molecules which guide a nuclease (Cas9) to its target DNA site. Upon mutation of the nuclease activity (dCas), dCas can be fused to transcriptional effectors. Such Artificial Transcription Factors (ATFs) can modulate the expression of any gene at will, although the effects are presumed to be transient. To induce sustained gene expression modulation, we exploit these DNA binding platforms to target epigenetic effector domains to genomic loci of interest. 

Re-expression of a tumour suppressor gene generally is effective and induces apoptosis. Repression of gene expression can also easily be obtained. Guidelines on how to stably interfere with epigenetic gene regulation, however, are currently largely lacking. These data demonstrate feasibility of long-term gene repression as well as re-expression. Epigenetic Editing thus opens novel therapeutic avenues, realising the curable genome concept. 

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10:15-10:30 Discussion

10:30-11:00 Coffee

11:00-11:30 Expanding the programmability of DNA recognition

Professor Daniel Summerer, Technical University Dortmund, Germany

Abstract

The recent discovery of oxidized 5-methylcytosine (mC) derivatives in mammalian genomes and their involvement in key biological processes has created a need for methods that allow for their simple analysis at user-defined genomic positions. 

The group has recently started to explore the potential of transcription-activator-like effectors (TALEs) as programmable DNA-binding receptors for the analysis of 5mC and its derivatives. TALEs recognize DNA via the major groove that contains unique chemical information for each of the nucleobases and thus offer an ‘expanded programmability of DNA recognition’. Professor Summerer here reports on the engineering of new nucleobase selectivities into TALEs, and their use for epigenetic nucleobase analysis in genomes by affinity enrichment. These studies establish TALEs as the only receptor molecules capable of a direct isolation of user-defined epigenetic DNA biomarkers from genomes. 

11:30-11:45 Discussion

11:45-12:15 The chemistry, structure and function of modified DNA bases

Sir Shankar Balasubramanian FMedSci FRS, University of Cambridge, UK

Abstract

It is now evident that there are numerous chemical modifications that occur naturally in DNA across species. These modifications can be incorporated and removed by natural enzymes and they can alter the structural and functional properties of the genome. Sir Shankar will discuss some recent studies that developed chemical methodologies to detect and decode modified bases in the genome and the applications of such methodologies to elucidate their roles in biology.

12:15-12:30 Discussion

13:30-14:00 Protein methyltransferase inhibitors as precision cancer therapeutics

Professor Robert A. Copeland, Epizyme, Inc, USA

Abstract

The protein methyltransferases (PMTs) constitute a large class of enzymes that catalyse the methylation of lysine or arginine residues on histones and other proteins. A number of PMTs have been shown to be genetically altered in cancers through, for example, gene amplification, chromosomal translocations and point mutations. The group is approaching drug discovery efforts against these enzymes in two distinct ways. First, they are approaching the PMTs as a target class, taking advantage of common aspects of their enzymology to develop a broad platform for small molecule inhibitor discovery. The second approach targets specific enzymes that are genetically altered in cancer, and for which strong evidence of an oncogenic role of enzymatic activity has been established. To date, the group has advanced small molecule inhibitors against three PMT targets to human clinical trials: pinometostat, an inhibitor of DOT1L for MLL-rearranged leukaemia; tazemetostat, an inhibitor of EZH2 for non-Hodgkin’s lymphoma, INI1-negative solid tumours and mesothelioma; and EPZ015938/GSK3326595, an inhibitor of PRMT5 for non-Hodgkin’s lymphoma and solid tumours.  Preclinical and clinical data for these investigational drugs will be presented.

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14:00-14:15 Discussion

14:15-14:30 Shifting the balance: epigenetic modulators in drug development

Dr Tamara Maes, Oryzon Genomics S.A., Spain

Abstract

Post-translational modifications of histones are closely associated with changes in transcription. Changes in histone lysine methyl marks are among the most prominent changes, and are mediated by methyltransferases and demethylases. Many diseases are characterised by transcriptional imbalances, and interference with epigenetic targets can be used to modulate these aberrant profiles. Here Dr Maes will report on the advances in the development of ORY-1001/RG6016, a potent selective inhibitor of LSD1, for oncology; and of ORY-2001, a dual inhibitor of LSD1 and MAO-B, for the treatment of neurodegenerative diseases. 

LSD1 inhibition compromises the leukaemic stem cell capacity in AML, and drives differentiation of blasts towards a more mature phenotype. Using a chemical probe for LSD1, it was initially shown that MLL translocated cells exhibit special sensitivity to LSD1 inhibition. Treating AML cells with the potent selective LSD1 inhibitor ORY-1001, the group confirmed responses at subnanomolar to nanomolar concentrations, and revealed that phenotypic changes were accompanied by a shift of the gene expression partially rebalancing the transcriptional profile towards that of normal monocytes/macrophages. The group has developed an activity based LSD1 chemoprobe and used it to pull down the protein and to unravel its network of interacting factors in AML. ORY-1001 has recently finalised a Phase I/IIa trial in relapsed or refractory acute leukaemia and these data corroborate the in vivo potency of ORY-1001 as an AML differentiating agent. LSD1 inhibitors are also highly active in SCLC, and ORY-1001/RG6016 is currently in a Phase I trial in this indication.

The potential role of LSD1 as a drug target is not limited to cancer. LSD1 is expressed in the brain and has a dual role in neuronal stem cell proliferation and neuronal differentiation / neurite extension. Using a proprietary chemoprobe based target engagement assay, the group has shown that they can modulate LSD1 activity in the brain using the brain penetrant dual inhibitor ORY-2001. Treatment with ORY-2001 in SAMP8 mice, a model for accelerated ageing and Alzheimer’s disease, rescues memory in the novel object recognition test. Again, it was found that phenotypic changes were accompanied by a shift and partial restoration of gene expression patterns in the hippocampus. A Phase I trial with ORY-2001 to assess the compounds’ tolerability, pharmacokinetics and pharmacodynamics in healthy young and elderly volunteers is nearing finalisation.

14:45-15:00 Discussion

15:00-15:30 Tea

15:30-16:00 Selectivity: design and serendipity for epigenetic targets

Dr Chun-wa Chung, GlaxoSmithKline, UK

Abstract

The family of bromodomain-containing proteins is an emerging class of epigenetic regulators that act as readers of the ‘histone code’ via recognition of proteins that have been specifically acetylated by histone acetyltransferases. Fuelled by the publication, in 2010, of high affinity inhibitors of the BET bromodomain subfamily of protein that have diverse therapeutic potential, this protein family has become an intensive area of research in industry and academia. Over 200 papers have been published since 2016, and BET compounds are now in clinical trials for oncology. However, despite this impressive demonstration of the potential of the BET subfamily as drug targets the function of other family members remain poorly understood.

To help to understand the potential of other bromodomains the group has developed chemical probes for use in cellular biology studies. Some properties of good chemical probes (potency, selectivity and cell permeability) may need to be equivalent to or even better than drugs, so their discovery can present significant challenges. This presentation will include the structural, computational and medicinal chemistry optimisation of fragment hits to chemical probes for the bromodomains of BRPF1 and ATAD2. How selectivity is assessed for multidomain complexes and the broader experience of using chemical probes for target validation will also be discussed.

16:00-16:15 Discussion

16:15-16:45 Assessing sirtuins as drug targets in human illnesses

Professor Antonello Mai, Sapienza University of Rome, Italy

Abstract

NAD⁺-dependent histone deacetylases (sirtuins, SIRT1-7) have emerged as potential therapeutic targets for treatment of human illnesses such as cancer, metabolic, cardiovascular and neurodegenerative diseases, and other age-related disorders. In Professor Mai's lab, chemically different series of sirtuin inhibitors (SIRTi) and activators (SIRTa) have been identified so far.

Among SIRTi, some sirtinol analogues, obtained by replacement of the benzamide linkage of the prototype with other bioisosteric groups, have been described to induce apoptosis and/or cytodifferentiation in human leukaemia U937 cells. One of them, salermide, was well tolerated by mice and prompted tumour-specific apoptosis in a wide range of human cancer cell lines. In addition, the group designed some (thio)barbituric acid analogues (BDF4s) whose prototype, MC2141, displayed in U937 cells high apoptosis induction and showed antiproliferative effects against cancer cells including cancer stem cells. More recently, the gorup reported a series of highly specific SIRT2 inhibitors based on the 1,2,4-oxadiazole scaffold, inducing apoptosis and/or antiproliferative effects in leukaemia.

In contrast to the number of SIRTi, only few SIRT activators are known. A number of 1,4-dihydropyridines (DHPs) were described by us as SIRT1 activators able to increase nitric oxide levels in human keratynocyte HaCat cells, and to ameliorate skin repair in a mouse model of wound healing. In addition, the group identified, some pyrrolo[1,2-a]quinoxalines as the first synthetic SIRT6 activators. Biochemical assays show direct binding to the SIRT6 catalytic core and potent activation of SIRT6-dependent deacetylation. Crystal structures of SIRT6/activator complexes reveal that the compounds bind to a SIRT6-specific acyl channel pocket and identify key interactions. These results establish potent SIRT6 activation with small molecules and provide a structural basis for further development of SIRT6 activators as tools and therapeutics.

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16:45-17:00 Discussion