Publications by authors named "Tom D Heightman"

51 Publications

Fragment-based drug discovery: opportunities for organic synthesis.

RSC Med Chem 2020 Dec 24;12(3):321-329. Epub 2020 Dec 24.

Astex Pharmaceuticals 436 Cambridge Science Park Cambridge CB4 0QA UK

This Review describes the increasing demand for organic synthesis to facilitate fragment-based drug discovery (FBDD), focusing on polar, unprotected fragments. In FBDD, X-ray crystal structures are used to design target molecules for synthesis with new groups added onto a fragment specific growth vectors. This requires challenging synthesis which slows down drug discovery, and some fragments are not progressed into optimisation due to synthetic intractability. We have evaluated the output from Astex's fragment screenings for a number of programs, including urokinase-type plasminogen activator, hematopoietic prostaglandin D2 synthase, and hepatitis C virus NS3 protease-helicase, and identified fragments that were not elaborated due, in part, to a lack of commercially available analogues and/or suitable synthetic methodology. This represents an opportunity for the development of new synthetic research to enable rapid access to novel chemical space and fragment optimisation.
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http://dx.doi.org/10.1039/d0md00375aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8130625PMC
December 2020

Structure-Activity and Structure-Conformation Relationships of Aryl Propionic Acid Inhibitors of the Kelch-like ECH-Associated Protein 1/Nuclear Factor Erythroid 2-Related Factor 2 (KEAP1/NRF2) Protein-Protein Interaction.

J Med Chem 2019 05 25;62(9):4683-4702. Epub 2019 Apr 25.

Astex Pharmaceuticals , 436 Cambridge Science Park , Cambridge CB4 0QA , U.K.

The KEAP1-NRF2-mediated cytoprotective response plays a key role in cellular homoeostasis. Insufficient NRF2 signaling during chronic oxidative stress may be associated with the pathophysiology of several diseases with an inflammatory component, and pathway activation through direct modulation of the KEAP1-NRF2 protein-protein interaction is being increasingly explored as a potential therapeutic strategy. Nevertheless, the physicochemical nature of the KEAP1-NRF2 interface suggests that achieving high affinity for a cell-penetrant druglike inhibitor might be challenging. We recently reported the discovery of a highly potent tool compound which was used to probe the biology associated with directly disrupting the interaction of NRF2 with the KEAP1 Kelch domain. We now present a detailed account of the medicinal chemistry campaign leading to this molecule, which included exploration and optimization of protein-ligand interactions in three energetic "hot spots" identified by fragment screening. In particular, we also discuss how consideration of ligand conformational stabilization was important to its development and present evidence for preorganization of the lead compound which may contribute to its high affinity and cellular activity.
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http://dx.doi.org/10.1021/acs.jmedchem.9b00279DOI Listing
May 2019

Enabling synthesis in fragment-based drug discovery by reactivity mapping: photoredox-mediated cross-dehydrogenative heteroarylation of cyclic amines.

Chem Sci 2019 Feb 21;10(8):2264-2271. Epub 2018 Dec 21.

Astex Pharmaceuticals , 436 Cambridge Science Park, Milton Road , Cambridge , CB4 0QA , UK . Email: ; Email:

In fragment-based drug discovery (FBDD), a weakly binding fragment hit is elaborated into a potent ligand by bespoke functionalization along specific directions (growth vectors) from the fragment core in order to complement the 3D structure of the target protein. This structure-based design approach can present significant synthetic challenges, as growth vectors often originate on sp or sp ring carbons which are not the most synthetically accessible points on the fragment. To address this issue and expedite synthesis in FBDD, we established a nanogram-to-gram workflow for the development of enabling synthetic transformations, such as the direct C-H functionalization of heterocycles. This novel approach deploys high-throughput experimentation (HTE) in 1536-well microtiter plates (MTPs) facilitated by liquid handling robots to screen reaction conditions on the nanomolar scale; subsequently the reaction is upscaled in a continuous flow to generate gram-quantities of the material. In this paper, we disclose the use of this powerful workflow for the development of a photoredox-mediated cross-dehydrogenative coupling of fragments and medicinally relevant heterocyclic precursors Minisci-type addition of α-amino radicals to electron-deficient heteroarenes. The optimized reaction conditions were employed on the milligram-scale on a diverse set of 112 substrates to map out structure-reactivity relationships (SRRs) of the transformation. The coupling exhibits excellent tolerance to a variety of functional groups and N-rich heteroarenes relevant to FBDD and was upscaled in a continuous flow to afford gram-quantities of pharmaceutically relevant sp-sp privileged architectures.
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http://dx.doi.org/10.1039/c8sc04789hDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6385880PMC
February 2019

Quantitation of ERK1/2 inhibitor cellular target occupancies with a reversible slow off-rate probe.

Chem Sci 2018 Dec 17;9(45):8608-8618. Epub 2018 Sep 17.

Astex Pharmaceuticals , 436 Cambridge Science Park , Cambridge , CB4 0QA , UK . Email: ; Email:

Target engagement is a key concept in drug discovery and its direct measurement can provide a quantitative understanding of drug efficacy and/or toxicity. Failure to demonstrate target occupancy in relevant cells and tissues has been recognised as a contributing factor to the low success rate of clinical drug development. Several techniques are emerging to quantify target engagement in cells; however, measurements remain challenging, mainly due to technical limitations. Here, we report the development of a non-covalent clickable probe, based on SCH772984, a slow off-rate ERK1/2 inhibitor, which enabled efficient pull down of ERK1/2 protein click reaction with tetrazine tagged agarose beads. This was used in a competition setting to measure relative target occupancy by selected ERK1/2 inhibitors. As a reference we used the cellular thermal shift assay, a label-free biophysical assay relying solely on ligand-induced thermodynamic stabilization of proteins. To validate the EC values measured by both methods, the results were compared with IC data for the phosphorylation of RSK, a downstream substrate of ERK1/2 used as a functional biomarker of ERK1/2 inhibition. We showed that a slow off-rate reversible probe can be used to efficiently pull down cellular proteins, significantly extending the potential of the approach beyond the need for covalent or photoaffinity warheads.
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http://dx.doi.org/10.1039/c8sc02754dDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6253716PMC
December 2018

Highly Potent Clickable Probe for Cellular Imaging of MDM2 and Assessing Dynamic Responses to MDM2-p53 Inhibition.

Bioconjug Chem 2018 06 12;29(6):2100-2106. Epub 2018 Jun 12.

Astex Pharmaceuticals , 436 Cambridge Science Park , Cambridge CB4 0QA , United Kingdom.

MDM2 is a key negative regulator of the p53 tumor suppressor. Direct binding of MDM2 to p53 represses the protein's transcriptional activity and induces its polyubiquitination, targeting it for degradation by the proteasome. Consequently, small molecule inhibitors that antagonize MDM2-p53 binding, such as RG7388, have progressed into clinical development aiming to reactivate p53 function in TP53 wild-type tumors. Here, we describe the design, synthesis, and biological evaluation of a trans-cyclooctene tagged derivative of RG7388, RG7388-TCO, which showed high cellular potency and specificity for MDM2. The in-cell reaction of RG7388-TCO with a tetrazine-tagged BODIPY dye enabled fluorescence imaging of endogenous MDM2 in SJSA-1 and T778 tumor cells. RG7388-TCO was also used to pull down MDM2 by reaction with tetrazine-tagged agarose beads in SJSA-1 lysates. The data presented show that RG733-TCO enables precise imaging of MDM2 in cells and can permit a relative assessment of target engagement and MDM2-p53 antagonism in vitro.
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http://dx.doi.org/10.1021/acs.bioconjchem.8b00315DOI Listing
June 2018

Fragment-Based Discovery of a Potent, Orally Bioavailable Inhibitor That Modulates the Phosphorylation and Catalytic Activity of ERK1/2.

J Med Chem 2018 06 31;61(11):4978-4992. Epub 2018 May 31.

Astex Pharmaceuticals , 436 Cambridge Science Park , Cambridge , CB4 0QA , U.K.

Aberrant activation of the MAPK pathway drives cell proliferation in multiple cancers. Inhibitors of BRAF and MEK kinases are approved for the treatment of BRAF mutant melanoma, but resistance frequently emerges, often mediated by increased signaling through ERK1/2. Here, we describe the fragment-based generation of ERK1/2 inhibitors that block catalytic phosphorylation of downstream substrates such as RSK but also modulate phosphorylation of ERK1/2 by MEK without directly inhibiting MEK. X-ray crystallographic and biophysical fragment screening followed by structure-guided optimization and growth from the hinge into a pocket proximal to the C-α helix afforded highly potent ERK1/2 inhibitors with excellent kinome selectivity. In BRAF mutant cells, the lead compound suppresses pRSK and pERK levels and inhibits proliferation at low nanomolar concentrations. The lead exhibits tumor regression upon oral dosing in BRAF mutant xenograft models, providing a promising basis for further optimization toward clinical pERK1/2 modulating ERK1/2 inhibitors.
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http://dx.doi.org/10.1021/acs.jmedchem.8b00421DOI Listing
June 2018

Protein degradation: a validated therapeutic strategy with exciting prospects.

Essays Biochem 2017 11 8;61(5):517-527. Epub 2017 Nov 8.

Astex Pharmaceuticals, 436 Cambridge Science Park, Cambridge CB4 0QA, U.K.

In a time of unprecedented challenges in developing potent, selective and well-tolerated protein inhibitors as therapeutics, drug hunters are increasingly seeking alternative modalities to modulate pharmacological targets. Selective inhibitors are achievable for only a fraction of the proteome, and are not guaranteed to elicit the desired response in patients, especially when pursuing targets identified through genetic knockdown. Targeted protein degradation holds the potential to expand the range of proteins that can be effectively modulated. Drugs inducing protein degradation through misfolding or by modulating cereblon (CRBN) substrate recognition are already approved for treatment of cancer patients. The last decade has seen the development of proteolysis targeting chimeras (PROTACs), small molecules that elicit proteasomal degradation by causing protein polyubiquitination. These have been used to degrade a range of disease-relevant proteins in cells, and some show promising efficacy in preclinical animal models, although their clinical efficacy and tolerability is yet to be proven. This review introduces current strategies for protein degradation with an emphasis on PROTACs and the role of click chemistry in PROTAC research through the formation of libraries of preclicked PROTACs or in-cell click-formed PROTACs (CLIPTACs).
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http://dx.doi.org/10.1042/EBC20170030DOI Listing
November 2017

Discovery of a Potent Nonpeptidomimetic, Small-Molecule Antagonist of Cellular Inhibitor of Apoptosis Protein 1 (cIAP1) and X-Linked Inhibitor of Apoptosis Protein (XIAP).

J Med Chem 2017 06 24;60(11):4611-4625. Epub 2017 May 24.

Astex Pharmaceuticals , 436 Cambridge Science Park, Milton Road, Cambridge CB4 0QA, U.K.

XIAP and cIAP1 are members of the inhibitor of apoptosis protein (IAP) family and are key regulators of anti-apoptotic and pro-survival signaling pathways. Overexpression of IAPs occurs in various cancers and has been associated with tumor progression and resistance to treatment. Structure-based drug design (SBDD) guided by structural information from X-ray crystallography, computational studies, and NMR solution conformational analysis was successfully applied to a fragment-derived lead resulting in AT-IAP, a potent, orally bioavailable, dual antagonist of XIAP and cIAP1 and a structurally novel chemical probe for IAP biology.
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http://dx.doi.org/10.1021/acs.jmedchem.6b01877DOI Listing
June 2017

Visualization of Endogenous ERK1/2 in Cells with a Bioorthogonal Covalent Probe.

Bioconjug Chem 2017 06 12;28(6):1677-1683. Epub 2017 May 12.

Astex Pharmaceuticals , 436 Cambridge Science Park, Cambridge CB4 0QA, U.K.

The RAS-RAF-MEK-ERK pathway has been intensively studied in oncology, with RAS known to be mutated in ∼30% of all human cancers. The recent emergence of ERK1/2 inhibitors and their ongoing clinical investigation demands a better understanding of ERK1/2 behavior following small-molecule inhibition. Although fluorescent fusion proteins and fluorescent antibodies are well-established methods of visualizing proteins, we show that ERK1/2 can be visualized via a less-invasive approach based on a two-step process using inverse electron demand Diels-Alder cycloaddition. Our previously reported trans-cyclooctene-tagged covalent ERK1/2 inhibitor was used in a series of imaging experiments following a click reaction with a tetrazine-tagged fluorescent dye. Although limitations were encountered with this approach, endogenous ERK1/2 was successfully imaged in cells, and "on-target" staining was confirmed by over-expressing DUSP5, a nuclear ERK1/2 phosphatase that anchors ERK1/2 in the nucleus.
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http://dx.doi.org/10.1021/acs.bioconjchem.7b00152DOI Listing
June 2017

Protein Degradation by In-Cell Self-Assembly of Proteolysis Targeting Chimeras.

ACS Cent Sci 2016 Dec 5;2(12):927-934. Epub 2016 Dec 5.

Astex Pharmaceuticals , 436 Cambridge Science Park, Cambridge, CB4 0QA, U.K.

Selective degradation of proteins by proteolysis targeting chimeras (PROTACs) offers a promising potential alternative to protein inhibition for therapeutic intervention. Current PROTAC molecules incorporate a ligand for the target protein, a linker, and an E3 ubiquitin ligase recruiting group, which bring together target protein and ubiquitinating machinery. Such hetero-bifunctional molecules require significant linker optimization and possess high molecular weight, which can limit cellular permeation, solubility, and other drug-like properties. We show here that the hetero-bifunctional molecule can be formed intracellularly by bio-orthogonal click combination of two smaller precursors. We designed a tetrazine tagged thalidomide derivative which reacts rapidly with a -cyclo-octene tagged ligand of the target protein in cells to form a cereblon E3 ligase recruiting PROTAC molecule. The in-cell click-formed proteolysis targeting chimeras (CLIPTACs) were successfully used to degrade two key oncology targets, BRD4 and ERK1/2. ERK1/2 degradation was achieved using a CLIPTAC based on a covalent inhibitor. We expect this approach to be readily extendable to other inhibitor-protein systems because the tagged E3 ligase recruiter is capable of undergoing the click reaction with a suitably tagged ligand of any protein of interest to elicit its degradation.
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http://dx.doi.org/10.1021/acscentsci.6b00280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5200928PMC
December 2016

Structure of the Epigenetic Oncogene MMSET and Inhibition by N-Alkyl Sinefungin Derivatives.

ACS Chem Biol 2016 11 27;11(11):3093-3105. Epub 2016 Sep 27.

Astex Pharmaceuticals , 436 Cambridge Science Park, Milton Road, Cambridge, United Kingdom CB4 0QA.

The members of the NSD subfamily of lysine methyl transferases are compelling oncology targets due to the recent characterization of gain-of-function mutations and translocations in several hematological cancers. To date, these proteins have proven intractable to small molecule inhibition. Here, we present initial efforts to identify inhibitors of MMSET (aka NSD2 or WHSC1) using solution phase and crystal structural methods. On the basis of 2D NMR experiments comparing NSD1 and MMSET structural mobility, we designed an MMSET construct with five point mutations in the N-terminal helix of its SET domain for crystallization experiments and elucidated the structure of the mutant MMSET SET domain at 2.1 Å resolution. Both NSD1 and MMSET crystal systems proved resistant to soaking or cocrystallography with inhibitors. However, use of the close homologue SETD2 as a structural surrogate supported the design and characterization of N-alkyl sinefungin derivatives, which showed low micromolar inhibition against both SETD2 and MMSET.
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http://dx.doi.org/10.1021/acschembio.6b00308DOI Listing
November 2016

In-gel activity-based protein profiling of a clickable covalent ERK1/2 inhibitor.

Mol Biosyst 2016 08;12(9):2867-74

Astex Pharmaceuticals, 436 Cambridge Science Park, Cambridge, CB4 0QA, UK.

In-gel activity-based protein profiling (ABPP) offers rapid assessment of the proteome-wide selectivity and target engagement of a chemical tool. Here we demonstrate the use of the inverse electron demand Diels Alder (IEDDA) click reaction for in-gel ABPP by evaluating the selectivity profile and target engagement of a covalent ERK1/2 probe tagged with a trans-cyclooctene group. The chemical probe was shown to bind covalently to Cys166 of ERK2 using protein MS and X-ray crystallography, and displayed submicromolar GI50s in A375 and HCT116 cells. In both cell lines, the probe demonstrated target engagement and a good selectivity profile at low concentrations, which was lost at higher concentrations. The IEDDA cycloaddition enabled fast and quantitative fluorescent tagging for readout with a high background-to-noise ratio and thereby provides a promising alternative to the commonly used copper catalysed alkyne-azide cycloaddition.
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http://dx.doi.org/10.1039/c6mb00367bDOI Listing
August 2016

Isoxazole-Derived Amino Acids are Bromodomain-Binding Acetyl-Lysine Mimics: Incorporation into Histone H4 Peptides and Histone H3.

Angew Chem Int Ed Engl 2016 07 6;55(29):8353-7. Epub 2016 Jun 6.

Department of Chemistry, Chemistry Research Laboratory, University of Oxford, Mansfield Road, Oxford, OX1 3TA, UK.

A range of isoxazole-containing amino acids was synthesized that displaced acetyl-lysine-containing peptides from the BAZ2A, BRD4(1), and BRD9 bromodomains. Three of these amino acids were incorporated into a histone H4-mimicking peptide and their affinity for BRD4(1) was assessed. Affinities of the isoxazole-containing peptides are comparable to those of a hyperacetylated histone H4-mimicking cognate peptide, and demonstrated a dependence on the position at which the unnatural residue was incorporated. An isoxazole-based alkylating agent was developed to selectively alkylate cysteine residues in situ. Selective monoalkylation of a histone H4-mimicking peptide, containing a lysine to cysteine residue substitution (K12C), resulted in acetyl-lysine mimic incorporation, with high affinity for the BRD4 bromodomain. The same technology was used to alkylate a K18C mutant of histone H3.
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http://dx.doi.org/10.1002/anie.201602908DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5089653PMC
July 2016

Monoacidic Inhibitors of the Kelch-like ECH-Associated Protein 1: Nuclear Factor Erythroid 2-Related Factor 2 (KEAP1:NRF2) Protein-Protein Interaction with High Cell Potency Identified by Fragment-Based Discovery.

J Med Chem 2016 04 12;59(8):3991-4006. Epub 2016 Apr 12.

GlaxoSmithKline Pharmaceuticals, 709 Swedeland Road, King of Prussia, Pennsylvania 19406, United States.

KEAP1 is the key regulator of the NRF2-mediated cytoprotective response, and increasingly recognized as a target for diseases involving oxidative stress. Pharmacological intervention has focused on molecules that decrease NRF2-ubiquitination through covalent modification of KEAP1 cysteine residues, but such electrophilic compounds lack selectivity and may be associated with off-target toxicity. We report here the first use of a fragment-based approach to directly target the KEAP1 Kelch-NRF2 interaction. X-ray crystallographic screening identified three distinct "hot-spots" for fragment binding within the NRF2 binding pocket of KEAP1, allowing progression of a weak fragment hit to molecules with nanomolar affinity for KEAP1 while maintaining drug-like properties. This work resulted in a promising lead compound which exhibits tight and selective binding to KEAP1, and activates the NRF2 antioxidant response in cellular and in vivo models, thereby providing a high quality chemical probe to explore the therapeutic potential of disrupting the KEAP1-NRF2 interaction.
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http://dx.doi.org/10.1021/acs.jmedchem.6b00228DOI Listing
April 2016

Optimization of sphingosine-1-phosphate-1 receptor agonists: effects of acidic, basic, and zwitterionic chemotypes on pharmacokinetic and pharmacodynamic profiles.

J Med Chem 2014 Dec 15;57(24):10424-42. Epub 2014 Dec 15.

Neurology Center of Excellence for Drug Discovery, GlaxoSmithKline , New Frontiers Science Park, Harlow CM19 5AW, U.K.

The efficacy of the recently approved drug fingolimod (FTY720) in multiple sclerosis patients results from the action of its phosphate metabolite on sphingosine-1-phosphate S1P1 receptors, while a variety of side effects have been ascribed to its S1P3 receptor activity. Although S1P and phospho-fingolimod share the same structural elements of a zwitterionic headgroup and lipophilic tail, a variety of chemotypes have been found to show S1P1 receptor agonism. Here we describe a study of the tolerance of the S1P1 and S1P3 receptors toward bicyclic heterocycles of systematically varied shape and connectivity incorporating acidic, basic, or zwitterionic headgroups. We compare their physicochemical properties, their performance in in vitro and in vivo pharmacokinetic models, and their efficacy in peripheral lymphocyte lowering. The campaign resulted in the identification of several potent S1P1 receptor agonists with good selectivity vs S1P3 receptors, efficacy at <1 mg/kg oral doses, and developability properties suitable for progression into preclinical development.
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http://dx.doi.org/10.1021/jm5010336DOI Listing
December 2014

BRD4 assists elongation of both coding and enhancer RNAs by interacting with acetylated histones.

Nat Struct Mol Biol 2014 Dec 10;21(12):1047-57. Epub 2014 Nov 10.

Program in Genomics of Differentiation, National Institutes of Child Health and Human Development, Bethesda, Maryland, USA.

Small-molecule BET inhibitors interfere with the epigenetic interactions between acetylated histones and the bromodomains of the BET family proteins, including BRD4, and they potently inhibit growth of malignant cells by targeting cancer-promoting genes. BRD4 interacts with the pause-release factor P-TEFb and has been proposed to release RNA polymerase II (Pol II) from promoter-proximal pausing. We show that BRD4 occupies widespread genomic regions in mouse cells and directly stimulates elongation of both protein-coding transcripts and noncoding enhancer RNAs (eRNAs), in a manner dependent on bromodomain function. BRD4 interacts with elongating Pol II complexes and assists Pol II in progression through hyperacetylated nucleosomes by interacting with acetylated histones via bromodomains. On active enhancers, the BET inhibitor JQ1 antagonizes BRD4-associated eRNA synthesis. Thus, BRD4 is involved in multiple steps of the transcription hierarchy, primarily by facilitating transcript elongation both at enhancers and on gene bodies independently of P-TEFb.
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http://dx.doi.org/10.1038/nsmb.2912DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4720983PMC
December 2014

Fragment-based discovery of 6-azaindazoles as inhibitors of bacterial DNA ligase.

ACS Med Chem Lett 2013 Dec 18;4(12):1208-12. Epub 2013 Oct 18.

Astex Pharmaceuticals Inc., 436 Cambridge Science Park, Milton Road, Cambridge CB4 0QA, United Kingdom.

Herein we describe the application of fragment-based drug design to bacterial DNA ligase. X-ray crystallography was used to guide structure-based optimization of a fragment-screening hit to give novel, nanomolar, AMP-competitive inhibitors. The lead compound 13 showed antibacterial activity across a range of pathogens. Data to demonstrate mode of action was provided using a strain of S. aureus, engineered to overexpress DNA ligase.
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http://dx.doi.org/10.1021/ml4003277DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4027552PMC
December 2013

A series of potent CREBBP bromodomain ligands reveals an induced-fit pocket stabilized by a cation-π interaction.

Angew Chem Int Ed Engl 2014 Jun 12;53(24):6126-30. Epub 2014 May 12.

Department of Chemistry, Chemistry Research Laboratory, University of Oxford, Mansfield Road, Oxford, OX1 3TA (UK) http://conway.chem.ox.ac.uk/

The benzoxazinone and dihydroquinoxalinone fragments were employed as novel acetyl lysine mimics in the development of CREBBP bromodomain ligands. While the benzoxazinone series showed low affinity for the CREBBP bromodomain, expansion of the dihydroquinoxalinone series resulted in the first potent inhibitors of a bromodomain outside the BET family. Structural and computational studies reveal that an internal hydrogen bond stabilizes the protein-bound conformation of the dihydroquinoxalinone series. The side chain of this series binds in an induced-fit pocket forming a cation-π interaction with R1173 of CREBBP. The most potent compound inhibits binding of CREBBP to chromatin in U2OS cells.
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http://dx.doi.org/10.1002/anie.201402750DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4298791PMC
June 2014

The discovery of the benzazepine class of histamine H3 receptor antagonists.

Bioorg Med Chem Lett 2013 Dec 5;23(24):6897-901. Epub 2013 Oct 5.

GlaxoSmithKline R&D, Neurology CEDD, Harlow, Essex CM19 5AW, United Kingdom. Electronic address:

This Letter describes the discovery of a novel series of H3 receptor antagonists. The initial medicinal chemistry strategy focused on deconstructing and simplifying an early screening hit which rapidly led to the discovery of a novel series of H3 receptor antagonists based on the benzazepine core. Employing an H3 driven pharmacodynamic model, the series was then further optimised through to a lead compound that showed robust in vivo functional activity and possessed overall excellent developability properties.
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http://dx.doi.org/10.1016/j.bmcl.2013.09.089DOI Listing
December 2013

WHSC1 links transcription elongation to HIRA-mediated histone H3.3 deposition.

EMBO J 2013 Aug 6;32(17):2392-406. Epub 2013 Aug 6.

Program in Genomics of Differentiation, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.

Actively transcribed genes are enriched with the histone variant H3.3. Although H3.3 deposition has been linked to transcription, mechanisms controlling this process remain elusive. We investigated the role of the histone methyltransferase Wolf-Hirschhorn syndrome candidate 1 (WHSC1) (NSD2/MMSET) in H3.3 deposition into interferon (IFN) response genes. IFN treatment triggered robust H3.3 incorporation into activated genes, which continued even after cessation of transcription. Likewise, UV radiation caused H3.3 deposition in UV-activated genes. However, in Whsc1(-/-) cells IFN- or UV-triggered H3.3 deposition was absent, along with a marked reduction in IFN- or UV-induced transcription. We found that WHSC1 interacted with the bromodomain protein 4 (BRD4) and the positive transcription elongation factor b (P-TEFb) and facilitated transcriptional elongation. WHSC1 also associated with HIRA, the H3.3-specific histone chaperone, independent of BRD4 and P-TEFb. WHSC1 and HIRA co-occupied IFN-stimulated genes and supported prolonged H3.3 incorporation, leaving a lasting transcriptional mark. Our results reveal a previously unrecognized role of WHSC1, which links transcriptional elongation and H3.3 deposition into activated genes through two molecularly distinct pathways.
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http://dx.doi.org/10.1038/emboj.2013.176DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3770338PMC
August 2013

BRD4 coordinates recruitment of pause release factor P-TEFb and the pausing complex NELF/DSIF to regulate transcription elongation of interferon-stimulated genes.

Mol Cell Biol 2013 Jun 15;33(12):2497-507. Epub 2013 Apr 15.

Program in Genomics of Differentiation, NICHD, National Institutes of Health, Bethesda, Maryland, USA.

RNA polymerase II (Pol II) and the pausing complex, NELF and DSIF, are detected near the transcription start site (TSS) of many active and silent genes. Active transcription starts when the pause release factor P-TEFb is recruited to initiate productive elongation. However, the mechanism of P-TEFb recruitment and regulation of NELF/DSIF during transcription is not fully understood. We investigated this question in interferon (IFN)-stimulated transcription, focusing on BRD4, a BET family protein that interacts with P-TEFb. Besides P-TEFb, BRD4 binds to acetylated histones through the bromodomain. We found that BRD4 and P-TEFb, although not present prior to IFN treatment, were robustly recruited to IFN-stimulated genes (ISGs) after stimulation. Likewise, NELF and DSIF prior to stimulation were hardly detectable on ISGs, which were strongly recruited after IFN treatment. A shRNA-based knockdown assay of NELF revealed that it negatively regulates the passage of Pol II and DSIF across the ISGs during elongation, reducing total ISG transcript output. Analyses with a BRD4 small-molecule inhibitor showed that IFN-induced recruitment of P-TEFb and NELF/DSIF was under the control of BRD4. We suggest a model where BRD4 coordinates both positive and negative regulation of ISG elongation.
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http://dx.doi.org/10.1128/MCB.01180-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3700095PMC
June 2013

The design and synthesis of 5- and 6-isoxazolylbenzimidazoles as selective inhibitors of the BET bromodomains.

Medchemcomm 2013 Jan;4(1):140-144

Structural Genomics Consortium, Nuffield Department of Clinical Medicine, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford OX3 7DQ, UK.

Simple 1-substituted 5- and 6-isoxazolyl-benzimidazoles have been shown to be potent inhibitors of the BET bromodomains with selectivity over the related bromodomain of CBP. The reported inhibitors were prepared from simple starting materials in two steps followed by separation of the regioisomers or regioselectively in three steps.
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http://dx.doi.org/10.1039/C2MD20189EDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4678575PMC
January 2013

Chemical biology of lysine demethylases.

Authors:
Tom D Heightman

Curr Chem Genomics 2011 22;5(Suppl 1):62-71. Epub 2011 Aug 22.

Structural Genomics Consortium, Oxford University, Roosevelt Drive, Oxford OX3 7DQ, UK.

Abnormal levels of DNA methylation and/or histone modifications are observed in patients with a wide variety of chronic diseases. Methylation of lysines within histone tails is a key modification that contributes to increased gene expression or repression depending on the specific residue and degree of methylation, which is in turn controlled by the interplay of lysine methyl transferases and demethylases. Drugs that target these and other enzymes controlling chromatin modifications can modulate the expression of clusters of genes, potentially offering higher therapeutic efficacy than classical agents acting on downstream biochemical pathways that are susceptible to degeneracy. Lysine demethylases, first discovered in 2004, are the subject of increasing interest as therapeutic targets. This review provides an overview of recent findings implicating lysine demethylases in a range of therapeutic areas including oncology, immunoinflammation, metabolic disorders, neuroscience, virology and regenerative medicine, together with a summary of recent advances in structural biology and small molecule inhibitor discovery, supporting the tractability of the protein family for the development of selective druglike inhibitors.
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http://dx.doi.org/10.2174/1875397301005010062DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3178875PMC
November 2011

3,5-dimethylisoxazoles act as acetyl-lysine-mimetic bromodomain ligands.

J Med Chem 2011 Oct 6;54(19):6761-70. Epub 2011 Sep 6.

Nuffield Department of Clinical Medicine, Structural Genomics Consortium, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford OX3 7DQ, U.K.

Histone-lysine acetylation is a vital chromatin post-translational modification involved in the epigenetic regulation of gene transcription. Bromodomains bind acetylated lysines, acting as readers of the histone-acetylation code. Competitive inhibitors of this interaction have antiproliferative and anti-inflammatory properties. With 57 distinct bromodomains known, the discovery of subtype-selective inhibitors of the histone-bromodomain interaction is of great importance. We have identified the 3,5-dimethylisoxazole moiety as a novel acetyl-lysine bioisostere, which displaces acetylated histone-mimicking peptides from bromodomains. Using X-ray crystallographic analysis, we have determined the interactions responsible for the activity and selectivity of 4-substituted 3,5-dimethylisoxazoles against a selection of phylogenetically diverse bromodomains. By exploiting these interactions, we have developed compound 4d, which has IC(50) values of <5 μM for the bromodomain-containing proteins BRD2(1) and BRD4(1). These compounds are promising leads for the further development of selective probes for the bromodomain and extra C-terminal domain (BET) family and CREBBP bromodomains.
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http://dx.doi.org/10.1021/jm200640vDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3188285PMC
October 2011

Discovery of a brain-penetrant S1P₃-sparing direct agonist of the S1P₁ and S1P₅ receptors efficacious at low oral dose.

J Med Chem 2011 Oct 9;54(19):6724-33. Epub 2011 Sep 9.

Immuno Inflammation Center of Excellence for Drug Discovery, GlaxoSmithKline, Gunnels Wood Road, Stevenage SG1 2NY, United Kingdom.

2-Amino-2-(4-octylphenethyl)propane-1,3-diol 1 (fingolimod, FTY720) has been recently marketed in the United States for the treatment of patients with remitting relapsing multiple sclerosis (RRMS). Its efficacy has been primarily linked to the agonism on T cells of S1P(1), one of the five sphingosine 1-phosphate (S1P) G-protein-coupled receptors, while its cardiovascular side effects have been associated with activity at S1P(3). Emerging data suggest that the ability of this molecule to cross the blood-brain barrier and to interact with both S1P(1) and S1P(5) in the central nervous system (CNS) may contribute to its efficacy in treating patients with RRMS. We have recently disclosed the structure of an advanced, first generation S1P(3)-sparing S1P(1) agonist, a zwitterion with limited CNS exposure. In this Article, we highlight our strategy toward the identification of CNS-penetrant S1P(3)-sparing S1P(1) and S1P(5) agonists resulting in the discovery of 5-(3-{2-[2-hydroxy-1-(hydroxymethyl)ethyl]-5-methyl-1,2,3,4-tetrahydro-6-isoquinolinyl}-1,2,4-oxadiazol-5-yl)-2-[(1-methylethyl)oxy]benzonitrile 15. Its exceptional in vivo potency and good pharmacokinetic properties translate into a very low predicted therapeutic dose in human (<1 mg p.o. once daily).
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http://dx.doi.org/10.1021/jm200609tDOI Listing
October 2011

Bromodomain-peptide displacement assays for interactome mapping and inhibitor discovery.

Mol Biosyst 2011 Oct 1;7(10):2899-908. Epub 2011 Aug 1.

Structural Genomics Consortium, University of Oxford, Oxford OX3 7DQ, UK.

Histone lysine acetylation is a key component of epigenetic regulation of gene transcription. Bromodomains, found in histone acetyl transferases and other chromatin-associated proteins, bind selectively to acetylated lysines, acting as "readers" of the histone code, and have recently been shown to contain a druggable binding pocket. Here we report the development of high-throughput assays that quantify the binding of bromodomains to acetylated histone peptides. We have used these assays to screen for histone binding partners of as yet uncharacterized bromodomains, adding to current knowledge of the histone code and expanding the repertoire of assays for chemical probe discovery. We have also demonstrated that these assays can be used to detect small molecule binding from the very weak to the nanomolar range. This assay methodology is thereby anticipated to provide the basis both for broader interactome profiling and for small molecule inhibitor discovery.
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http://dx.doi.org/10.1039/c1mb05099kDOI Listing
October 2011

A selective inhibitor and probe of the cellular functions of Jumonji C domain-containing histone demethylases.

J Am Chem Soc 2011 Jun 31;133(24):9451-6. Epub 2011 May 31.

Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309, USA.

Histone methylations are important chromatin marks that regulate gene expression, genomic stability, DNA repair, and genomic imprinting. Histone demethylases are the most recent family of histone-modifying enzymes discovered. Here, we report the characterization of a small-molecule inhibitor of Jumonji C domain-containing histone demethylases. The inhibitor derives from a structure-based design and preferentially inhibits the subfamily of trimethyl lysine demethylases. Its methyl ester prodrug, methylstat, selectively inhibits Jumonji C domain-containing his-tone demethylases in cells and may be a useful small-molecule probe of chromatin and its role in epigenetics.
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http://dx.doi.org/10.1021/ja201597bDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3133600PMC
June 2011

Synthesis and Biological Evaluation of JAHAs: Ferrocene-Based Histone Deacetylase Inhibitors.

ACS Med Chem Lett 2011 May 18;2(5):358-362. Epub 2011 Mar 18.

N(1)-Hydroxy-N(8)-ferrocenyloctanediamide, JAHA (7), an organometallic analogue of SAHA containing a ferrocenyl group as a phenyl bioisostere, displays nanomolar inhibition of class I HDACs, excellent selectivity over class IIa HDACs, and anticancer action in intact cells (IC(50) = 2.4 μM, MCF7 cell line). Molecular docking studies of 7 in HDAC8 (a,b) suggested that the ferrocenyl moiety in 7 can overlap with the aryl cap of SAHA and should display similar HDAC inhibition, which was borne out in an in vitro assay (IC(50) values against HDAC8 (μM, SD in parentheses): SAHA, 1.41 (0.15); 7, 1.36 (0.16). Thereafter, a small library of related JAHA analogues has been synthesized, and preliminary SAR studies are presented. IC(50) values as low as 90 pM toward HDAC6 (class IIb) have been determined, highlighting the excellent potential of JAHAs as bioinorganic probes.
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http://dx.doi.org/10.1021/ml100295vDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3093745PMC
May 2011

Therapeutic prospects for epigenetic modulation.

Authors:
Tom D Heightman

Expert Opin Ther Targets 2011 Jun 3;15(6):729-40. Epub 2011 Mar 3.

Astex Therapeutics Ltd., 436 Cambridge Science Park, Cambridge CB4 0QA, UK.

Introduction: Epigenetics describes the phenomenon of heritable changes in gene regulation governed by non-Mendelian processes, primarily through biochemical modifications to chromatin that occur during cell differentiation and development. Abnormal levels of DNA and/or histone modifications are observed in patients with a wide variety of chronic diseases. Drugs that target the proteins controlling these chromatin modifications can modulate the expression of clusters of genes, potentially offering higher therapeutic efficacy than classical agents with single target pharmacologies that are susceptible to biochemical pathway degeneracy.

Areas Covered: This article reviews research characterizing dysregulation of epigenetic processes in cancer, immuno-inflammatory, psychiatric, neurological, metabolic and virology disease areas, and summarizes recent developments in identifying small molecule modulators that are being used to inform target discovery and initiate drug discovery projects.

Expert Opinion: There are numerous potential opportunities for epigenetic modulators in treating a wide range of chronic diseases; however, the field is complex, involving > 300 proteins, and much work is still required to provide tools to unravel the functions of individual proteins, particularly in vivo. This groundwork is essential to allow the drug discovery community to focus on those epigenetic proteins most likely to be suitable targets for safe, efficacious new therapies.
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http://dx.doi.org/10.1517/14728222.2011.561786DOI Listing
June 2011