Publications by authors named "Peter D Craggs"

16 Publications

  • Page 1 of 1

Reducing False Positives through the Application of Fluorescence Lifetime Technology: A Comparative Study Using TYK2 Kinase as a Model System.

SLAS Discov 2021 Mar 30:24725552211002472. Epub 2021 Mar 30.

Medicine Design, Medicinal Science and Technology, GlaxoSmithKline, Stevenage, Hertfordshire, UK.

The predominant assay detection methodologies used for enzyme inhibitor identification during early-stage drug discovery are fluorescence-based. Each fluorophore has a characteristic fluorescence decay, known as the fluorescence lifetime, that occurs throughout a nanosecond-to-millisecond timescale. The measurement of fluorescence lifetime as a reporter for biological activity is less common than fluorescence intensity, even though the latter has numerous issues that can lead to false-positive readouts. The confirmation of hit compounds as true inhibitors requires additional assays, cost, and time to progress from hit identification to lead drug-candidate optimization. To explore whether the use of fluorescence lifetime technology (FLT) can offer comparable benefits to label-free-based approaches such as RapidFire mass spectroscopy (RF-MS) and a superior readout compared to time-resolved fluorescence resonance energy transfer (TR-FRET), three equivalent assays were developed against the clinically validated tyrosine kinase 2 (TYK2) and screened against annotated compound sets. FLT provided a marked decrease in the number of false-positive hits when compared to TR-FRET. Further cellular screening confirmed that a number of potential inhibitors directly interacted with TYK2 and inhibited the downstream phosphorylation of the signal transducer and activator of transcription 4 protein (STAT4).
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http://dx.doi.org/10.1177/24725552211002472DOI Listing
March 2021

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

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

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

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

Cellular Target Engagement Approaches to Monitor Epigenetic Reader Domain Interactions.

SLAS Discov 2020 02 25;25(2):163-175. Epub 2019 Dec 25.

Medicine Design, Medicinal Science and Technology, GlaxoSmithKline, Stevenage, Hertfordshire, UK.

Malfunctions in the basic epigenetic mechanisms such as histone modifications, DNA methylation, and chromatin remodeling are implicated in a number of cancers and immunological and neurodegenerative conditions. Within GlaxoSmithKline (GSK) we have utilized a number of variations of the NanoBRET technology for the direct measurement of compound-target engagement within native cellular environments to drive high-throughput, routine structure-activity relationship (SAR) profiling across differing epigenetic targets. NanoBRET is a variation of the bioluminescence resonance energy transfer (BRET) methodology utilizing proteins of interest fused to either NanoLuc, a small, high-emission-intensity luciferase, or HaloTag, a modified dehalogenase enzyme that can be selectively labeled with a fluorophore. The combination of these two technologies has enabled the application of NanoBRET to biological systems such as epigenetic protein-protein interactions, which have previously been challenging. By synergizing target engagement assays with more complex primary cell phenotypic assays, we have been able to demonstrate compound-target selectivity profiles to enhance cellular potency and offset potential liability risks. Additionally, we have shown that in the absence of a robust, cell phenotypic assay, it is possible to utilize NanoBRET target engagement assays to aid chemistry in progressing at a higher scale than would have otherwise been achievable. The NanoBRET target engagement assays utilized have further shown an excellent correlation with more reductionist biochemical and biophysical assay systems, clearly demonstrating the possibility of using such assay systems at scale, in tandem with, or in preference to, lower-throughput cell phenotypic approaches.
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http://dx.doi.org/10.1177/2472555219896278DOI Listing
February 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

Identification of Novel Trypanosoma cruzi Proteasome Inhibitors Using a Luminescence-Based High-Throughput Screening Assay.

Antimicrob Agents Chemother 2019 09 23;63(9). Epub 2019 Aug 23.

Drug Discovery Unit, Wellcome Centre for Anti-Infectives Research, School of Life Sciences, University of Dundee, Dundee, United Kingdom

Chagas' disease, caused by the protozoan parasite , is a potentially life-threatening condition that has become a global issue. Current treatment is limited to two medicines that require prolonged dosing and are associated with multiple side effects, which often lead to treatment discontinuation and failure. One way to address these shortcomings is through target-based drug discovery on validated protein targets. One such target is the proteasome, which plays a crucial role in protein degradation and turnover through chymotrypsin-, trypsin-, and caspase-like catalytic activities. In order to initiate a proteasome drug discovery program, we isolated proteasomes from epimastigotes and characterized their activity using a commercially available glow-like luminescence-based assay. We developed a high-throughput biochemical assay for the chymotrypsin-like activity of the proteasome, which was found to be sensitive, specific, and robust but prone to luminescence technology interference. To mitigate this, we also developed a counterscreen assay that identifies potential interferers at the levels of both the luciferase enzyme reporter and the mechanism responsible for a glow-like response. Interestingly, we also found that the peptide substrate for chymotrypsin-like proteasome activity was not specific and was likely partially turned over by other catalytic sites of the protein. Finally, we utilized these biochemical tools to screen 18,098 compounds, exploring diverse drug-like chemical space, which allowed us to identify 39 hits that were active in the primary screening assay and inactive in the counterscreen assay.
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http://dx.doi.org/10.1128/AAC.00309-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6709497PMC
September 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

The Mechanism of Acetyl Transfer Catalyzed by Mycobacterium tuberculosis GlmU.

Biochemistry 2018 06 2;57(24):3387-3401. Epub 2018 May 2.

Platform Technology and Science , GlaxoSmithKline , Stevenage , U.K.

The biosynthetic pathway of peptidoglycan is essential for Mycobacterium tuberculosis. We report here the acetyltransferase substrate specificity and catalytic mechanism of the bifunctional N-acetyltransferase/uridylyltransferase from M. tuberculosis (GlmU). This enzyme is responsible for the final two steps of the synthesis of UDP- N-acetylglucosamine, which is an essential precursor of peptidoglycan, from glucosamine 1-phosphate, acetyl-coenzyme A, and uridine 5'-triphosphate. GlmU utilizes ternary complex formation to transfer an acetyl from acetyl-coenzyme A to glucosamine 1-phosphate to form N-acetylglucosamine 1-phosphate. Steady-state kinetic studies and equilibrium binding experiments indicate that GlmU follows a steady-state ordered kinetic mechanism, with acetyl-coenzyme A binding first, which triggers a conformational change in GlmU, followed by glucosamine 1-phosphate binding. Coenzyme A is the last product to dissociate. Chemistry is partially rate-limiting as indicated by pH-rate studies and solvent kinetic isotope effects. A novel crystal structure of a mimic of the Michaelis complex, with glucose 1-phosphate and acetyl-coenzyme A, helps us to propose the residues involved in deprotonation of glucosamine 1-phosphate and the loop movement that likely generates the active site required for glucosamine 1-phosphate to bind. Together, these results pave the way for the rational discovery of improved inhibitors against M. tuberculosis GlmU, some of which might become candidates for antibiotic discovery programs.
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http://dx.doi.org/10.1021/acs.biochem.8b00121DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6011181PMC
June 2018

Discovery of a Potent, Cell Penetrant, and Selective p300/CBP-Associated Factor (PCAF)/General Control Nonderepressible 5 (GCN5) Bromodomain Chemical Probe.

J Med Chem 2017 01 9;60(2):695-709. Epub 2017 Jan 9.

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

p300/CREB binding protein associated factor (PCAF/KAT2B) and general control nonderepressible 5 (GCN5/KAT2A) are multidomain proteins that have been implicated in retroviral infection, inflammation pathways, and cancer development. However, outside of viral replication, little is known about the dependence of these effects on the C-terminal bromodomain. Herein, we report GSK4027 as a chemical probe for the PCAF/GCN5 bromodomain, together with GSK4028 as an enantiomeric negative control. The probe was optimized from a weakly potent, nonselective pyridazinone hit to deliver high potency for the PCAF/GCN5 bromodomain, high solubility, cellular target engagement, and ≥18000-fold selectivity over the BET family, together with ≥70-fold selectivity over the wider bromodomain families.
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http://dx.doi.org/10.1021/acs.jmedchem.6b01566DOI Listing
January 2017

GSK6853, a Chemical Probe for Inhibition of the BRPF1 Bromodomain.

ACS Med Chem Lett 2016 Jun 9;7(6):552-7. Epub 2016 May 9.

Epinova Discovery Performance Unit, Quantitative Pharmacology, Experimental Medicine Unit, Flexible Discovery Unit, and Platform Technology and Science, GlaxoSmithKline , Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, U.K.

The BRPF (Bromodomain and PHD Finger-containing) protein family are important scaffolding proteins for assembly of MYST histone acetyltransferase complexes. A selective benzimidazolone BRPF1 inhibitor showing micromolar activity in a cellular target engagement assay was recently described. Herein, we report the optimization of this series leading to the identification of a superior BRPF1 inhibitor suitable for in vivo studies.
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http://dx.doi.org/10.1021/acsmedchemlett.6b00092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4904261PMC
June 2016

Fragment-Based Discovery of Low-Micromolar ATAD2 Bromodomain Inhibitors.

J Med Chem 2015 Jul 9;58(14):5649-73. Epub 2015 Jul 9.

∥Drug Metabolism and Pharmacokinetics (DMPK), GlaxoSmithKline, Park Road, Ware, Hertfordshire SG12 0DP, United Kingdom.

Overexpression of ATAD2 (ATPase family, AAA domain containing 2) has been linked to disease severity and progression in a wide range of cancers, and is implicated in the regulation of several drivers of cancer growth. Little is known of the dependence of these effects upon the ATAD2 bromodomain, which has been categorized as among the least tractable of its class. The absence of any potent, selective inhibitors limits clear understanding of the therapeutic potential of the bromodomain. Here, we describe the discovery of a hit from a fragment-based targeted array. Optimization of this produced the first known micromolar inhibitors of the ATAD2 bromodomain.
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http://dx.doi.org/10.1021/acs.jmedchem.5b00772DOI Listing
July 2015

Inhibition of PAD4 activity is sufficient to disrupt mouse and human NET formation.

Nat Chem Biol 2015 Mar 26;11(3):189-91. Epub 2015 Jan 26.

1] Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, Massachusetts, USA. [2] Division of Hematology/Oncology, Boston Children's Hospital, Boston, Massachusetts, USA. [3] Department of Pediatrics, Harvard Medical School, Boston, Massachusetts, USA.

PAD4 has been strongly implicated in the pathogenesis of autoimmune, cardiovascular and oncological diseases through clinical genetics and gene disruption in mice. New selective PAD4 inhibitors binding a calcium-deficient form of the PAD4 enzyme have validated the critical enzymatic role of human and mouse PAD4 in both histone citrullination and neutrophil extracellular trap formation for, to our knowledge, the first time. The therapeutic potential of PAD4 inhibitors can now be explored.
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http://dx.doi.org/10.1038/nchembio.1735DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4397581PMC
March 2015

1,3-Dimethyl Benzimidazolones Are Potent, Selective Inhibitors of the BRPF1 Bromodomain.

ACS Med Chem Lett 2014 Nov 10;5(11):1190-5. Epub 2014 Sep 10.

Epinova Discovery Performance Unit and Molecular Discovery Research, GlaxoSmithKline , Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, U.K.

The BRPF (bromodomain and PHD finger-containing) protein family are important scaffolding proteins for assembly of MYST histone acetyltransferase complexes. Here, we report the discovery, binding mode, and structure-activity relationship (SAR) of the first potent, selective series of inhibitors of the BRPF1 bromodomain.
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http://dx.doi.org/10.1021/ml5002932DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4233354PMC
November 2014

BET inhibition silences expression of MYCN and BCL2 and induces cytotoxicity in neuroblastoma tumor models.

PLoS One 2013 23;8(8):e72967. Epub 2013 Aug 23.

Cancer Epigenetics Discovery Performance Unit, GlaxoSmithKline, Collegeville, Pennsylvania, USA.

BET family proteins are epigenetic regulators known to control expression of genes involved in cell growth and oncogenesis. Selective inhibitors of BET proteins exhibit potent anti-proliferative activity in a number of hematologic cancer models, in part through suppression of the MYC oncogene and downstream Myc-driven pathways. However, little is currently known about the activity of BET inhibitors in solid tumor models, and whether down-regulation of MYC family genes contributes to sensitivity. Here we provide evidence for potent BET inhibitor activity in neuroblastoma, a pediatric solid tumor associated with a high frequency of MYCN amplifications. We treated a panel of neuroblastoma cell lines with a novel small molecule inhibitor of BET proteins, GSK1324726A (I-BET726), and observed potent growth inhibition and cytotoxicity in most cell lines irrespective of MYCN copy number or expression level. Gene expression analyses in neuroblastoma cell lines suggest a role of BET inhibition in apoptosis, signaling, and N-Myc-driven pathways, including the direct suppression of BCL2 and MYCN. Reversal of MYCN or BCL2 suppression reduces the potency of I-BET726-induced cytotoxicity in a cell line-specific manner; however, neither factor fully accounts for I-BET726 sensitivity. Oral administration of I-BET726 to mouse xenograft models of human neuroblastoma results in tumor growth inhibition and down-regulation MYCN and BCL2 expression, suggesting a potential role for these genes in tumor growth. Taken together, our data highlight the potential of BET inhibitors as novel therapeutics for neuroblastoma, and suggest that sensitivity is driven by pleiotropic effects on cell growth and apoptotic pathways in a context-specific manner.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0072967PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3751846PMC
April 2014