Publications by authors named "Chun-wa Chung"

86 Publications

The development of highly potent and selective small molecule correctors of Z α-antitrypsin misfolding.

Bioorg Med Chem Lett 2021 Jun 19;41:127973. Epub 2021 Mar 19.

UCL Respiratory, Rayne Institute, University College London, London WC1E 6JF, United Kingdom.

α1-antitrypsin deficiency is characterised by the misfolding and intracellular polymerisation of mutant α1-antitrypsin protein within the endoplasmic reticulum (ER) of hepatocytes. Small molecules that bind and stabilise Z α-antitrypsin were identified via a DNA-encoded library screen. A subsequent structure based optimisation led to a series of highly potent, selective and cellular active α1-antitrypsin correctors.
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http://dx.doi.org/10.1016/j.bmcl.2021.127973DOI Listing
June 2021

Template-Hopping Approach Leads to Potent, Selective, and Highly Soluble Bromo and Extraterminal Domain (BET) Second Bromodomain (BD2) Inhibitors.

J Med Chem 2021 Mar 4;64(6):3249-3281. Epub 2021 Mar 4.

Epigenetics Discovery Performance Unit, GlaxoSmithKline, Medicines Research Centre, Stevenage, Hertfordshire SG1 2NY, U.K.

A number of reports have recently been published describing the discovery and optimization of bromo and extraterminal inhibitors which are selective for the second bromodomain (BD2); these include our own work toward GSK046 () and GSK620 (). This paper describes our approach to mitigating the genotoxicity risk of GSK046 by replacement of the acetamide functionality with a heterocyclic ring. This was followed by a template-hopping and hybridization approach, guided by structure-based drug design, to incorporate learnings from other BD2-selective series, optimize the vector for the amide region, and explore the ZA cleft, leading to the identification of potent, selective, and bioavailable compounds (GSK452), (GSK737), and (GSK217).
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http://dx.doi.org/10.1021/acs.jmedchem.0c02156DOI Listing
March 2021

Half-Life Extension of BMP1/TLL Metalloproteinase Inhibitors Using Small-Molecule Human Serum Albumin Binders.

Bioconjug Chem 2021 02 1;32(2):279-289. Epub 2021 Feb 1.

GlaxoSmithKline US, 1250 South Collegeville Road, Collegeville, Pennsylvania 19426, United States.

Reducing the required frequence of drug dosing can improve the adherence of patients to chronic treatments. Hence, drugs with longer half-lives are highly desirable. One of the most promising approaches to extend the half-life of drugs is conjugation to human serum albumin (HSA). In this work, we describe the use of , a small-molecule noncovalent HSA binder, to extend the half-life and pharmacology of small-molecule BMP1/TLL inhibitors in humanized mice (HSA KI/KI). A series of conjugates of with BMP1/TLL inhibitors were prepared. In particular, showed good solubility and a half-life extension of >20-fold versus the parent molecule in the HSA KI/KI mice, reaching half-lives of >48 h with maintained maximal inhibition of plasma BMP1/TLL. The same conjugate showed a half-life of only 3 h in the wild-type mice, suggesting that the half-life extension was principally due to specific interactions with HSA. It is envisioned that conjugation to should be applicable to a wide range of small molecule or peptide drugs with short half-lives. In this context, AlbuBinders represent a viable alternative to existing half-life extension technologies.
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http://dx.doi.org/10.1021/acs.bioconjchem.0c00662DOI Listing
February 2021

Development of a small molecule that corrects misfolding and increases secretion of Z α -antitrypsin.

EMBO Mol Med 2021 Mar 29;13(3):e13167. Epub 2021 Jan 29.

GlaxoSmithKline, Cambridge, MA, USA.

Severe α -antitrypsin deficiency results from the Z allele (Glu342Lys) that causes the accumulation of homopolymers of mutant α -antitrypsin within the endoplasmic reticulum of hepatocytes in association with liver disease. We have used a DNA-encoded chemical library to undertake a high-throughput screen to identify small molecules that bind to, and stabilise Z α -antitrypsin. The lead compound blocks Z α -antitrypsin polymerisation in vitro, reduces intracellular polymerisation and increases the secretion of Z α -antitrypsin threefold in an iPSC model of disease. Crystallographic and biophysical analyses demonstrate that GSK716 and related molecules bind to a cryptic binding pocket, negate the local effects of the Z mutation and stabilise the bound state against progression along the polymerisation pathway. Oral dosing of transgenic mice at 100 mg/kg three times a day for 20 days increased the secretion of Z α -antitrypsin into the plasma by sevenfold. There was no observable clearance of hepatic inclusions with respect to controls over the same time period. This study provides proof of principle that "mutation ameliorating" small molecules can block the aberrant polymerisation that underlies Z α -antitrypsin deficiency.
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http://dx.doi.org/10.15252/emmm.202013167DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7933930PMC
March 2021

GSK973 Is an Inhibitor of the Second Bromodomains (BD2s) of the Bromodomain and Extra-Terminal (BET) Family.

ACS Med Chem Lett 2020 Aug 6;11(8):1581-1587. Epub 2020 Jul 6.

Epigenetics Discovery Performance Unit and Platform Technology and Science, GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, U.K.

Pan-BET inhibitors have shown profound efficacy in a number of in vivo preclinical models and have entered the clinic in oncology trials where adverse events have been reported. These inhibitors interact equipotently with the eight bromodomains of the BET family of proteins. To better understand the contribution of each domain to their efficacy and to improve from their safety profile, selective inhibitors are required. This Letter discloses the profile of GSK973, a highly selective inhibitor of the second bromodomains of the BET proteins that has undergone extensive preclinical in vitro and in vivo characterization.
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http://dx.doi.org/10.1021/acsmedchemlett.0c00247DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7429977PMC
August 2020

Structure-Based Design of a Bromodomain and Extraterminal Domain (BET) Inhibitor Selective for the N-Terminal Bromodomains That Retains an Anti-inflammatory and Antiproliferative Phenotype.

J Med Chem 2020 09 3;63(17):9020-9044. Epub 2020 Aug 3.

GSK, Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, United Kingdom.

The bromodomain and extraterminal domain (BET) family of epigenetic regulators comprises four proteins (BRD2, BRD3, BRD4, BRDT), each containing tandem bromodomains. To date, small molecule inhibitors of these proteins typically bind all eight bromodomains of the family with similar affinity, resulting in a diverse range of biological effects. To enable further understanding of the broad phenotype characteristic of pan-BET inhibition, the development of inhibitors selective for individual, or sets of, bromodomains within the family is required. In this regard, we report the discovery of a potent probe molecule possessing up to 150-fold selectivity for the N-terminal bromodomains (BD1s) over the C-terminal bromodomains (BD2s) of the BETs. Guided by structural information, a specific amino acid difference between BD1 and BD2 domains was targeted for selective interaction with chemical functionality appended to the previously developed I-BET151 scaffold. Data presented herein demonstrate that selective inhibition of BD1 domains is sufficient to drive anti-inflammatory and antiproliferative effects.
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http://dx.doi.org/10.1021/acs.jmedchem.0c00566DOI Listing
September 2020

A Photoaffinity-Based Fragment-Screening Platform for Efficient Identification of Protein Ligands.

Angew Chem Int Ed Engl 2020 11 7;59(47):21096-21105. Epub 2020 Sep 7.

GlaxoSmithKline, Gunnels Wood Road, Stevenage, Hertfordshire, SG1 2NY, UK.

Advances in genomic analyses enable the identification of new proteins that are associated with disease. To validate these targets, tool molecules are required to demonstrate that a ligand can have a disease-modifying effect. Currently, as tools are reported for only a fraction of the proteome, platforms for ligand discovery are essential to leverage insights from genomic analyses. Fragment screening offers an efficient approach to explore chemical space. Presented here is a fragment-screening platform, termed PhABits (PhotoAffinity Bits), which utilizes a library of photoreactive fragments to covalently capture fragment-protein interactions. Hits can be profiled to determine potency and the site of crosslinking, and subsequently developed as reporters in a competitive displacement assay to identify novel hit matter. The PhABit platform is envisioned to be widely applicable to novel protein targets, identifying starting points in the development of therapeutics.
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http://dx.doi.org/10.1002/anie.202008361DOI Listing
November 2020

The Optimization of a Novel, Weak Bromo and Extra Terminal Domain (BET) Bromodomain Fragment Ligand to a Potent and Selective Second Bromodomain (BD2) Inhibitor.

J Med Chem 2020 09 30;63(17):9093-9126. Epub 2020 Aug 30.

IVIVT Cellzome, Platform Technology and Science, GlaxoSmithKline, Meyerhofstrasse 1, 69117 Heidelberg, Germany.

The profound efficacy, yet associated toxicity of pan-BET inhibitors is well documented. The possibility of an ameliorated safety profile driven by significantly selective (>100-fold) inhibition of a subset of the eight bromodomains is enticing, but challenging given the close homology. Herein, we describe the X-ray crystal structure-directed optimization of a novel weak fragment ligand with a pan-second bromodomain (BD2) bias, to potent and highly BD2 selective inhibitors. A template hopping approach, enabled by our parallel research into an orthogonal template (, GSK046), was the basis for the high selectivity observed. This culminated in two tool molecules, (GSK620) and (GSK549), which showed an anti-inflammatory phenotype in human whole blood, confirming their cellular target engagement. Excellent broad selectivity, developability, and in vivo oral pharmacokinetics characterize these tools, which we hope will be of broad utility to the field of epigenetics research.
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http://dx.doi.org/10.1021/acs.jmedchem.0c00796DOI Listing
September 2020

Structural Insights into PROTAC-Mediated Degradation of Bcl-xL.

ACS Chem Biol 2020 09 13;15(9):2316-2323. Epub 2020 Aug 13.

Protein Degradation Group, GlaxoSmithKline, Gunnels Wood Road, Stevenage, SG1 2NY, United Kingdom.

The Bcl-2 family of proteins, such as Bcl-xL and Bcl-2, play key roles in cancer cell survival. Structural studies of Bcl-xL formed the foundation for the development of the first Bcl-2 family inhibitors and FDA approved drugs. Recently, teolysis rgeting himeras (PROTACs) that degrade Bcl-xL have been proposed as a therapeutic modality with the potential to enhance potency and reduce toxicity versus antagonists. However, no ternary complex structures of Bcl-xL with a PROTAC and an E3 ligase have been successfully determined to guide this approach. Herein, we report the design, characterization, and X-ray structure of a VHL E3 ligase-recruiting Bcl-xL PROTAC degrader. The 1.9 Å heterotetrameric structure, composed of (ElonginB:ElonginC:VHL):PROTAC:Bcl-xL, reveals an extensive network of neo-interactions, between the E3 ligase and the target protein, and between noncognate parts of the PROTAC and partner proteins. This work illustrates the challenges associated with the rational design of bifunctional molecules where interactions involve composite interfaces.
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http://dx.doi.org/10.1021/acschembio.0c00266DOI Listing
September 2020

Design and Synthesis of a Highly Selective and -Capable Inhibitor of the Second Bromodomain of the Bromodomain and Extra Terminal Domain Family of Proteins.

J Med Chem 2020 09 20;63(17):9070-9092. Epub 2020 Aug 20.

Epigenetics Discovery Performance Unit, GlaxoSmithKline, Medicines Research Centre, Stevenage, Hertfordshire SG1 2NY, U.K.

Pan-bromodomain and extra terminal domain (BET) inhibitors interact equipotently with the eight bromodomains of the BET family of proteins and have shown profound efficacy in a number of phenotypic assays and pre-clinical models in inflammation or oncology. A number of these inhibitors have progressed to the clinic where pharmacology-driven adverse events have been reported. To better understand the contribution of each domain to their efficacy and improve their safety profile, selective inhibitors are required. This article discloses the profile of GSK046, also known as iBET-BD2, a highly selective inhibitor of the second bromodomains of the BET proteins that has undergone extensive pre-clinical and characterization.
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http://dx.doi.org/10.1021/acs.jmedchem.0c00605DOI Listing
September 2020

GSK789: A Selective Inhibitor of the First Bromodomains (BD1) of the Bromo and Extra Terminal Domain (BET) Proteins.

J Med Chem 2020 09 4;63(17):9045-9069. Epub 2020 Aug 4.

Molecular Discovery Research, GlaxoSmithKline, Cellzome GmbH, Meyerhofstrasse 1, 69117 Heidelberg, Germany.

Pan-bromodomain and extra terminal (BET) inhibitors interact equipotently with all eight bromodomains of the BET family of proteins. They have shown profound efficacy in vitro and in vivo in oncology and immunomodulatory models, and a number of them are currently in clinical trials where significant safety signals have been reported. It is therefore important to understand the functional contribution of each bromodomain to assess the opportunity to tease apart efficacy and toxicity. This article discloses the in vitro and cellular activity profiles of GSK789, a potent, cell-permeable, and highly selective inhibitor of the first bromodomains of the BET family.
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http://dx.doi.org/10.1021/acs.jmedchem.0c00614DOI Listing
September 2020

Design and Development of a Macrocyclic Series Targeting Phosphoinositide 3-Kinase δ.

ACS Med Chem Lett 2020 Jul 3;11(7):1386-1391. Epub 2020 Jun 3.

GSK Medicines Research Centre, Gunnels Wood Road, Stevenage SG1 2NY, United Kingdom.

A macrocyclization approach has been explored on a series of benzoxazine phosphoinositide 3-kinase δ inhibitors, resulting in compounds with improved potency, permeability, and clearance while maintaining good solubility. The thermodynamics of binding was explored via surface plasmon resonance, and the binding of lead macrocycle was found to be almost exclusively entropically driven compared with progenitor , which demonstrated both enthalpic and entropic contributions. The pharmacokinetics of macrocycle was also explored , where it showed reduced clearance when compared with the progenitor . This work adds to the growing body of evidence that macrocyclization could provide an alternative and complementary approach to the design of small-molecule inhibitors, with the potential to deliver differentiated properties.
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http://dx.doi.org/10.1021/acsmedchemlett.0c00061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7357223PMC
July 2020

Application of Atypical Acetyl-lysine Methyl Mimetics in the Development of Selective Inhibitors of the Bromodomain-Containing Protein 7 (BRD7)/Bromodomain-Containing Protein 9 (BRD9) Bromodomains.

J Med Chem 2020 06 29;63(11):5816-5840. Epub 2020 May 29.

GlaxoSmithKline R&D, Stevenage SG1 2NY, Hertfordshire, United Kingdom.

Non-BET bromodomain-containing proteins have become attractive targets for the development of novel therapeutics targeting epigenetic pathways. To help facilitate the target validation of this class of proteins, structurally diverse small-molecule ligands and methodologies to produce selective inhibitors in a predictable fashion are in high demand. Herein, we report the development and application of atypical acetyl-lysine (KAc) methyl mimetics to take advantage of the differential stability of conserved water molecules in the bromodomain binding site. Discovery of the -butyl group as an atypical KAc methyl mimetic allowed generation of (GSK6776) as a soluble, permeable, and selective BRD7/9 inhibitor from a pyridazinone template. The -butyl group was then used to enhance the bromodomain selectivity of an existing BRD9 inhibitor and to transform pan-bromodomain inhibitors into BRD7/9 selective compounds. Finally, a solvent-exposed vector was defined from the pyridazinone template to enable bifunctional molecule synthesis, and affinity enrichment chemoproteomic experiments were used to confirm several of the endogenous protein partners of BRD7 and BRD9, which form part of the chromatin remodeling PBAF and BAF complexes, respectively.
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http://dx.doi.org/10.1021/acs.jmedchem.0c00075DOI Listing
June 2020

Optimization of Potent ATAD2 and CECR2 Bromodomain Inhibitors with an Atypical Binding Mode.

J Med Chem 2020 05 6;63(10):5212-5241. Epub 2020 May 6.

WestCHEM, Department of Pure and Applied Chemistry, University of Strathclyde, Thomas Graham Building, 295 Cathedral Street, Glasgow G1 1XL, United Kingdom.

Most bromodomain inhibitors mimic the interactions of the natural acetylated lysine (KAc) histone substrate through key interactions with conserved asparagine and tyrosine residues within the binding pocket. Herein we report the optimization of a series of phenyl sulfonamides that exhibit a novel mode of binding to non-bromodomain and extra terminal domain (non-BET) bromodomains through displacement of a normally conserved network of four water molecules. Starting from an initial hit molecule, we report its divergent optimization toward the ATPase family AAA domain containing 2 (ATAD2) and cat eye syndrome chromosome region, candidate 2 (CECR2) domains. This work concludes with the identification of (GSK232), a highly selective, cellularly penetrant CECR2 inhibitor with excellent physicochemical properties.
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http://dx.doi.org/10.1021/acs.jmedchem.0c00021DOI Listing
May 2020

Exploring the SAR of the β-Ketoacyl-ACP Synthase Inhibitor GSK3011724A and Optimization around a Genotoxic Metabolite.

ACS Infect Dis 2020 05 20;6(5):1098-1109. Epub 2020 Mar 20.

Global Health R&D, GlaxoSmithKline, Severo Ochoa 2, Tres Cantos, 28760 Madrid, Spain.

In the course of optimizing a novel indazole sulfonamide series that inhibits β-ketoacyl-ACP synthase (KasA) of , a mutagenic aniline metabolite was identified. Further lead optimization efforts were therefore dedicated to eliminating this critical liability by removing the embedded aniline moiety or modifying its steric or electronic environment. While the narrow SAR space against the target ultimately rendered this goal unsuccessful, key structural knowledge around the binding site of this underexplored target for TB was generated to inform future discovery efforts.
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http://dx.doi.org/10.1021/acsinfecdis.9b00493DOI Listing
May 2020

Selective targeting of BD1 and BD2 of the BET proteins in cancer and immunoinflammation.

Science 2020 04 19;368(6489):387-394. Epub 2020 Mar 19.

Cellzome GmbH, Functional Genomics R&D, GlaxoSmithKline, Heidelberg, Germany.

The two tandem bromodomains of the BET (bromodomain and extraterminal domain) proteins enable chromatin binding to facilitate transcription. Drugs that inhibit both bromodomains equally have shown efficacy in certain malignant and inflammatory conditions. To explore the individual functional contributions of the first (BD1) and second (BD2) bromodomains in biology and therapy, we developed selective BD1 and BD2 inhibitors. We found that steady-state gene expression primarily requires BD1, whereas the rapid increase of gene expression induced by inflammatory stimuli requires both BD1 and BD2 of all BET proteins. BD1 inhibitors phenocopied the effects of pan-BET inhibitors in cancer models, whereas BD2 inhibitors were predominantly effective in models of inflammatory and autoimmune disease. These insights into the differential requirement of BD1 and BD2 for the maintenance and induction of gene expression may guide future BET-targeted therapies.
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http://dx.doi.org/10.1126/science.aaz8455DOI Listing
April 2020

Discovery of a Bromodomain and Extraterminal Inhibitor with a Low Predicted Human Dose through Synergistic Use of Encoded Library Technology and Fragment Screening.

J Med Chem 2020 01 6;63(2):714-746. Epub 2020 Jan 6.

GSK , Gunnels Wood Road , Stevenage , Hertfordshire SG1 2NY , U.K.

The bromodomain and extraterminal (BET) family of bromodomain-containing proteins are important regulators of the epigenome through their ability to recognize -acetyl lysine (KAc) post-translational modifications on histone tails. These interactions have been implicated in various disease states and, consequently, disruption of BET-KAc binding has emerged as an attractive therapeutic strategy with a number of small molecule inhibitors now under investigation in the clinic. However, until the utility of these advanced candidates is fully assessed by these trials, there remains scope for the discovery of inhibitors from new chemotypes with alternative physicochemical, pharmacokinetic, and pharmacodynamic profiles. Herein, we describe the discovery of a candidate-quality dimethylpyridone benzimidazole compound which originated from the hybridization of a dimethylphenol benzimidazole series, identified using encoded library technology, with an -methyl pyridone series identified through fragment screening. Optimization via structure- and property-based design led to I-BET469, which possesses favorable oral pharmacokinetic properties, displays activity in vivo, and is projected to have a low human efficacious dose.
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http://dx.doi.org/10.1021/acs.jmedchem.9b01670DOI Listing
January 2020

Identification of Selective Inhibitors of N-Myristoyltransferase by High-Throughput Screening.

J Med Chem 2020 01 8;63(2):591-600. Epub 2020 Jan 8.

Center for Infectious Disease Research , Seattle , Washington 98109 , United States.

New drugs that target species, the causative agents of malaria, are needed. The enzyme -myristoyltransferase (NMT) is an essential protein, which catalyzes the myristoylation of protein substrates, often to mediate membrane targeting. We screened ∼1.8 million small molecules for activity against () NMT. Hits were triaged based on potency and physicochemical properties and further tested against and () NMTs. We assessed the activity of hits against human NMT1 and NMT2 and discarded compounds with low selectivity indices. We identified 23 chemical classes specific for the inhibition of NMTs over human NMTs, including multiple novel scaffolds. Cocrystallization of NMT with one compound revealed peptide binding pocket binding. Other compounds show a range of potential modes of action. Our data provide insight into the activity of a collection of selective inhibitors of NMT and serve as a starting point for subsequent medicinal chemistry efforts.
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http://dx.doi.org/10.1021/acs.jmedchem.9b01343DOI Listing
January 2020

Single-Domain Antibodies as Crystallization Chaperones to Enable Structure-Based Inhibitor Development for RBR E3 Ubiquitin Ligases.

Cell Chem Biol 2020 01 5;27(1):83-93.e9. Epub 2019 Dec 5.

Molecular Structure of Cell Signalling Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK. Electronic address:

Protein ubiquitination plays a key role in the regulation of cellular processes, and misregulation of the ubiquitin system is linked to many diseases. So far, development of tool compounds that target enzymes of the ubiquitin system has been slow and only a few specific inhibitors are available. Here, we report the selection of single-domain antibodies (single-dAbs) based on a human scaffold that recognize the catalytic domain of HOIP, a subunit of the multi-component E3 LUBAC and member of the RBR family of E3 ligases. Some of these dAbs affect ligase activity and provide mechanistic insight into the ubiquitin transfer mechanism of different E2-conjugating enzymes. Furthermore, we show that the co-crystal structure of a HOIP RBR/dAb complex serves as a robust platform for soaking of ligands that target the active site cysteine of HOIP, thereby providing easy access to structure-based ligand design for this important class of E3 ligases.
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http://dx.doi.org/10.1016/j.chembiol.2019.11.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6963773PMC
January 2020

A Qualified Success: Discovery of a New Series of ATAD2 Bromodomain Inhibitors with a Novel Binding Mode Using High-Throughput Screening and Hit Qualification.

J Med Chem 2019 08 9;62(16):7506-7525. Epub 2019 Aug 9.

GlaxoSmithKline Tres Cantos , 28760 Tres Cantos , Madrid , Spain.

The bromodomain of ATAD2 has proved to be one of the least-tractable proteins within this target class. Here, we describe the discovery of a new class of inhibitors by high-throughput screening and show how the difficulties encountered in establishing a screening triage capable of finding progressible hits were overcome by data-driven optimization. Despite the prevalence of nonspecific hits and an exceptionally low progressible hit rate (0.001%), our optimized hit qualification strategy employing orthogonal biophysical methods enabled us to identify a single active series. The compounds have a novel ATAD2 binding mode with noncanonical features including the displacement of all conserved water molecules within the active site and a halogen-bonding interaction. In addition to reporting this new series and preliminary structure-activity relationship, we demonstrate the value of diversity screening to complement the knowledge-based approach used in our previous ATAD2 work. We also exemplify tactics that can increase the chance of success when seeking new chemical starting points for novel and less-tractable targets.
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http://dx.doi.org/10.1021/acs.jmedchem.9b00673DOI Listing
August 2019

Structure-Guided Identification of Resistance Breaking Antimalarial N‑Myristoyltransferase Inhibitors.

Cell Chem Biol 2019 07 9;26(7):991-1000.e7. Epub 2019 May 9.

Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK. Electronic address:

The attachment of myristate to the N-terminal glycine of certain proteins is largely a co-translational modification catalyzed by N-myristoyltransferase (NMT), and involved in protein membrane-localization. Pathogen NMT is a validated therapeutic target in numerous infectious diseases including malaria. In Plasmodium falciparum, NMT substrates are important in essential processes including parasite gliding motility and host cell invasion. Here, we generated parasites resistant to a particular NMT inhibitor series and show that resistance in an in vitro parasite growth assay is mediated by a single amino acid substitution in the NMT substrate-binding pocket. The basis of resistance was validated and analyzed with a structure-guided approach using crystallography, in combination with enzyme activity, stability, and surface plasmon resonance assays, allowing identification of another inhibitor series unaffected by this substitution. We suggest that resistance studies incorporated early in the drug development process help selection of drug combinations to impede rapid evolution of parasite resistance.
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http://dx.doi.org/10.1016/j.chembiol.2019.03.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6658617PMC
July 2019

Preclinical candidate for the treatment of visceral leishmaniasis that acts through proteasome inhibition.

Proc Natl Acad Sci U S A 2019 05 8;116(19):9318-9323. Epub 2019 Apr 8.

Drug Discovery Unit, Wellcome Centre for Anti-Infectives Research, Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee, Dundee DD1 5EH, United Kingdom.

Visceral leishmaniasis (VL), caused by the protozoan parasites and , is one of the major parasitic diseases worldwide. There is an urgent need for new drugs to treat VL, because current therapies are unfit for purpose in a resource-poor setting. Here, we describe the development of a preclinical drug candidate, GSK3494245/DDD01305143/compound 8, with potential to treat this neglected tropical disease. The compound series was discovered by repurposing hits from a screen against the related parasite Subsequent optimization of the chemical series resulted in the development of a potent cidal compound with activity against a range of clinically relevant and isolates. Compound 8 demonstrates promising pharmacokinetic properties and impressive in vivo efficacy in our mouse model of infection comparable with those of the current oral antileishmanial miltefosine. Detailed mode of action studies confirm that this compound acts principally by inhibition of the chymotrypsin-like activity catalyzed by the β5 subunit of the proteasome. High-resolution cryo-EM structures of apo and compound 8-bound 20S proteasome reveal a previously undiscovered inhibitor site that lies between the β4 and β5 proteasome subunits. This induced pocket exploits β4 residues that are divergent between humans and kinetoplastid parasites and is consistent with all of our experimental and mutagenesis data. As a result of these comprehensive studies and due to a favorable developability and safety profile, compound 8 is being advanced toward human clinical trials.
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http://dx.doi.org/10.1073/pnas.1820175116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6511062PMC
May 2019

Fragment-Based Covalent Ligand Screening Enables Rapid Discovery of Inhibitors for the RBR E3 Ubiquitin Ligase HOIP.

J Am Chem Soc 2019 02 4;141(6):2703-2712. Epub 2019 Feb 4.

Molecular Structure of Cell Signalling Laboratory , The Francis Crick Institute , 1 Midland Road , London NW1 1AT , United Kingdom.

Modification of proteins with polyubiquitin chains is a key regulatory mechanism to control cellular behavior and alterations in the ubiquitin system are linked to many diseases. Linear (M1-linked) polyubiquitin chains play pivotal roles in several cellular signaling pathways mediating immune and inflammatory responses and apoptotic cell death. These chains are formed by the linear ubiquitin chain assembly complex (LUBAC), a multiprotein E3 ligase that consists of 3 subunits, HOIP, HOIL-1L, and SHARPIN. Herein, we describe the discovery of inhibitors targeting the active site cysteine of the catalytic subunit HOIP using fragment-based covalent ligand screening. We report the synthesis of a diverse library of electrophilic fragments and demonstrate an integrated use of protein LC-MS, biochemical ubiquitination assays, chemical synthesis, and protein crystallography to enable the first structure-based development of covalent inhibitors for an RBR E3 ligase. Furthermore, using cell-based assays and chemoproteomics, we demonstrate that these compounds effectively penetrate mammalian cells to label and inhibit HOIP and NF-κB activation, making them suitable hits for the development of selective probes to study LUBAC biology. Our results illustrate the power of fragment-based covalent ligand screening to discover lead compounds for challenging targets, which holds promise to be a general approach for the development of cell-permeable inhibitors of thioester-forming E3 ubiquitin ligases.
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http://dx.doi.org/10.1021/jacs.8b13193DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6383986PMC
February 2019

Cryo-EM in drug discovery.

Biochem Soc Trans 2019 02 15;47(1):281-293. Epub 2019 Jan 15.

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

The impact of structural biology on drug discovery is well documented, and the workhorse technique for the past 30 years or so has been X-ray crystallography. With the advent of several technological improvements, including direct electron detectors, automation, better microscope vacuums and lenses, phase plates and improvements in computing power enabled by GPUs, it is now possible to record and analyse images of protein structures containing high-resolution information. This review, from a pharmaceutical perspective, highlights some of the most relevant and interesting protein structures for the pharmaceutical industry and shows examples of how ligand-binding sites, membrane proteins, both big and small, pseudo symmetry and complexes are being addressed by this technique.
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http://dx.doi.org/10.1042/BST20180267DOI Listing
February 2019

Aiming to Miss a Moving Target: Bromo and Extra Terminal Domain (BET) Selectivity in Constrained ATAD2 Inhibitors.

J Med Chem 2018 09 18;61(18):8321-8336. Epub 2018 Sep 18.

Molecular Discovery Research, Cellzome GmbH , GlaxoSmithKline , Meyerhofstrasse 1 , 69117 Heidelberg , Germany.

ATAD2 is a cancer-associated protein whose bromodomain has been described as among the least druggable of its class. In our recent disclosure of the first chemical probe against this bromodomain, GSK8814 (6), we described the use of a conformationally constrained methoxy piperidine to gain selectivity over the BET bromodomains. Here we describe an orthogonal conformational restriction strategy of the piperidine ring to give potent and selective tropane inhibitors and show structural insights into why this was more challenging than expected. Greater understanding of why different rational approaches succeeded or failed should help in the future design of selectivity in the bromodomain family.
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http://dx.doi.org/10.1021/acs.jmedchem.8b00862DOI Listing
September 2018

Novel insight into the reaction of nitro, nitroso and hydroxylamino benzothiazinones and of benzoxacinones with Mycobacterium tuberculosis DprE1.

Sci Rep 2018 09 7;8(1):13473. Epub 2018 Sep 7.

Platform Technology & Science, GlaxoSmithKline, Gunnels Wood Road, Stevenage, SG1 2NY, United Kingdom.

Nitro-substituted 1,3-benzothiazinones (nitro-BTZs) are mechanism-based covalent inhibitors of Mycobacterium tuberculosis decaprenylphosphoryl-β-D-ribose-2'-oxidase (DprE1) with strong antimycobacterial properties. We prepared a number of oxidized and reduced forms of nitro-BTZs to probe the mechanism of inactivation of the enzyme and to identify opportunities for further chemistry. The kinetics of inactivation of DprE1 was examined using an enzymatic assay that monitored reaction progress up to 100 min, permitting compound ranking according to k/K values. The side-chain at the 2-position and heteroatom identity at the 1-position of the BTZs were found to be important for inhibitory activity. We obtained crystal structures with several compounds covalently bound. The data suggest that steps upstream from the covalent end-points are likely the key determinants of potency and reactivity. The results of protein mass spectrometry using a 7-chloro-nitro-BTZ suggest that nucleophilic reactions at the 7-position do not operate and support a previously proposed mechanism in which BTZ activation by a reduced flavin intermediate is required. Unexpectedly, a hydroxylamino-BTZ showed time-dependent inhibition and mass spectrometry corroborated that this hydroxylamino-BTZ is a mechanism-based suicide inhibitor of DprE1. With this BTZ derivative, we propose a new covalent mechanism of inhibition of DprE1 that takes advantage of the oxidation cycle of the enzyme.
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http://dx.doi.org/10.1038/s41598-018-31316-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6128881PMC
September 2018

Integration of Lead Discovery Tactics and the Evolution of the Lead Discovery Toolbox.

SLAS Discov 2018 10 6;23(9):881-897. Epub 2018 Jun 6.

1 GlaxoSmithKline Drug Design and Selection, Platform Technology and Science, Stevenage, Hertfordshire, UK.

There has been much debate around the success rates of various screening strategies to identify starting points for drug discovery. Although high-throughput target-based and phenotypic screening has been the focus of this debate, techniques such as fragment screening, virtual screening, and DNA-encoded library screening are also increasingly reported as a source of new chemical equity. Here, we provide examples in which integration of more than one screening approach has improved the campaign outcome and discuss how strengths and weaknesses of various methods can be used to build a complementary toolbox of approaches, giving researchers the greatest probability of successfully identifying leads. Among others, we highlight case studies for receptor-interacting serine/threonine-protein kinase 1 and the bromo- and extra-terminal domain family of bromodomains. In each example, the unique insight or chemistries individual approaches provided are described, emphasizing the synergy of information obtained from the various tactics employed and the particular question each tactic was employed to answer. We conclude with a short prospective discussing how screening strategies are evolving, what this screening toolbox might look like in the future, how to maximize success through integration of multiple tactics, and scenarios that drive selection of one combination of tactics over another.
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http://dx.doi.org/10.1177/2472555218778503DOI Listing
October 2018

Discovery of Tetrahydroquinoxalines as Bromodomain and Extra-Terminal Domain (BET) Inhibitors with Selectivity for the Second Bromodomain.

J Med Chem 2018 05 3;61(10):4317-4334. Epub 2018 May 3.

WestCHEM, Department of Pure and Applied Chemistry , University of Strathclyde, Thomas Graham Building , 295 Cathedral Street , Glasgow , G1 1XL , U.K.

The bromodomain and extra-terminal domain (BET) family of proteins bind acetylated lysine residues on histone proteins. The four BET bromodomains-BRD2, BRD3, BRD4, and BRDT-each contain two bromodomain modules. BET bromodomain inhibition is a potential therapy for various cancers and immunoinflammatory diseases, but few reported inhibitors show selectivity within the BET family. Inhibitors with selectivity for the first or second bromodomain are desired to aid investigation of the biological function of these domains. Focused library screening identified a series of tetrahydroquinoxalines with selectivity for the second bromodomains of the BET family (BD2). Structure-guided optimization of the template improved potency, selectivity, and physicochemical properties, culminating in potent BET inhibitors with BD2 selectivity.
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http://dx.doi.org/10.1021/acs.jmedchem.7b01666DOI Listing
May 2018