Publications by authors named "Andrew J Pope"

16 Publications

  • Page 1 of 1

Discovery and Development of TMPRSS6 Inhibitors Modulating Hepcidin Levels in Human Hepatocytes.

Cell Chem Biol 2019 11 19;26(11):1559-1572.e9. Epub 2019 Sep 19.

Department of Pharmacology-Physiology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, 3001, 12(e) Avenue Nord, Sherbrooke, QC J1H 5N4, Canada; Institut de Pharmacologie de Sherbrooke, Université de Sherbrooke, Sherbrooke, QC, Canada. Electronic address:

Iron overload disorders are characterized by the body's inability to regulate iron absorption and its storage which can lead to organ failures. Accumulated evidence has revealed that hepcidin, the master regulator of iron homeostasis, is negatively modulated by TMPRSS6 (matriptase-2), a liver-specific type II transmembrane serine protease (TTSP). Here, we report that treatment with a peptidomimetic inhibitor affecting TMPRSS6 activity increases hepcidin production in hepatic cells. Moreover, similar effects were observed when using non-peptidic inhibitors obtained through optimization of hits from high-throughput screening. Using HepG2 cells and human primary hepatocytes, we show that TMPRSS6 inhibitors block TMPRSS6-dependent hemojuvelin cleavage and increase HAMP expression and levels of secreted hepcidin.
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http://dx.doi.org/10.1016/j.chembiol.2019.09.004DOI Listing
November 2019

Identification via a Parallel Hit Progression Strategy of Improved Small Molecule Inhibitors of the Malaria Purine Uptake Transporter that Inhibit Parasite Proliferation.

ACS Infect Dis 2019 10 14;5(10):1738-1753. Epub 2019 Aug 14.

Platform Technology & Science and Discovery Partners in Academia , GlaxoSmithKline , 1250 South Collegeville Road , Collegeville , Pennsylvania 19426 , United States.

Emerging resistance to current antimalarial medicines underscores the importance of identifying new drug targets and novel compounds. Malaria parasites are purine auxotrophic and import purines via the equilibrative nucleoside transporter type 1 (PfENT1). We previously showed that PfENT1 inhibitors block parasite proliferation in culture. Our goal was to identify additional, possibly more optimal chemical starting points for a drug discovery campaign. We performed a high throughput screen (HTS) of GlaxoSmithKline's 1.8 million compound library with a yeast-based assay to identify PfENT1 inhibitors. We used a parallel progression strategy for hit validation and expansion, with an emphasis on chemical properties in addition to potency. In one arm, the most active hits were tested for human cell toxicity; 201 had minimal toxicity. The second arm, hit expansion, used a scaffold-based substructure search with the HTS hits as templates to identify over 2000 compounds; 123 compounds had activity. Of these 324 compounds, 175 compounds inhibited proliferation of parasite strain 3D7 with IC values between 0.8 and ∼180 μM. One hundred forty-two compounds inhibited PfENT1 knockout (Δ) parasite growth, indicating they also hit secondary targets. Thirty-two hits inhibited growth of 3D7 but not Δ parasites. Thus, PfENT1 inhibition was sufficient to block parasite proliferation. Therefore, PfENT1 may be a viable target for antimalarial drug development. Six compounds with novel chemical scaffolds were extensively characterized in yeast-, parasite-, and human-erythrocyte-based assays. The inhibitors showed similar potencies against drug sensitive and resistant strains. They represent attractive starting points for development of novel antimalarial drugs.
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http://dx.doi.org/10.1021/acsinfecdis.9b00168DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7171677PMC
October 2019

Inhibitors of LexA Autoproteolysis and the Bacterial SOS Response Discovered by an Academic-Industry Partnership.

ACS Infect Dis 2018 03 8;4(3):349-359. Epub 2018 Jan 8.

Department of Medicine, Department of Biochemistry and Biophysics , University of Pennsylvania , 3610 Hamilton Walk , Philadelphia , Pennsylvania 19104 , United States.

The RecA/LexA axis of the bacterial DNA damage (SOS) response is a promising, yet nontraditional, drug target. The SOS response is initiated upon genotoxic stress, when RecA, a DNA damage sensor, induces LexA, the SOS repressor, to undergo autoproteolysis, thereby derepressing downstream genes that can mediate DNA repair and accelerate mutagenesis. As genetic inhibition of the SOS response sensitizes bacteria to DNA damaging antibiotics and decreases acquired resistance, inhibitors of the RecA/LexA axis could potentiate our current antibiotic arsenal. Compounds targeting RecA, which has many mammalian homologues, have been reported; however, small-molecules targeting LexA autoproteolysis, a reaction unique to the prokaryotic SOS response, have remained elusive. Here, we describe the logistics and accomplishments of an academic-industry partnership formed to pursue inhibitors against the RecA/LexA axis. A novel fluorescence polarization assay reporting on RecA-induced self-cleavage of LexA enabled the screening of 1.8 million compounds. Follow-up studies on select leads show distinct activity patterns in orthogonal assays, including several with activity in cell-based assays reporting on SOS activation. Mechanistic assays demonstrate that we have identified first-in-class small molecules that specifically target the LexA autoproteolysis step in SOS activation. Our efforts establish a realistic example for navigating academic-industry partnerships in pursuit of anti-infective drugs and offer starting points for dedicated lead optimization of SOS inhibitors that could act as adjuvants for current antibiotics.
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http://dx.doi.org/10.1021/acsinfecdis.7b00122DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5893282PMC
March 2018

Prioritizing multiple therapeutic targets in parallel using automated DNA-encoded library screening.

Nat Commun 2017 07 17;8:16081. Epub 2017 Jul 17.

GlaxoSmithKline, 830 Winter Street, Waltham, Massachusetts 02451, USA.

The identification and prioritization of chemically tractable therapeutic targets is a significant challenge in the discovery of new medicines. We have developed a novel method that rapidly screens multiple proteins in parallel using DNA-encoded library technology (ELT). Initial efforts were focused on the efficient discovery of antibacterial leads against 119 targets from Acinetobacter baumannii and Staphylococcus aureus. The success of this effort led to the hypothesis that the relative number of ELT binders alone could be used to assess the ligandability of large sets of proteins. This concept was further explored by screening 42 targets from Mycobacterium tuberculosis. Active chemical series for six targets from our initial effort as well as three chemotypes for DHFR from M. tuberculosis are reported. The findings demonstrate that parallel ELT selections can be used to assess ligandability and highlight opportunities for successful lead and tool discovery.
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http://dx.doi.org/10.1038/ncomms16081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5520047PMC
July 2017

A High-Content Imaging Screen for Cellular Regulators of β-Catenin Protein Abundance.

J Biomol Screen 2016 Mar 9;21(3):260-8. Epub 2015 Dec 9.

R&D Platform Technology Sciences, GlaxoSmithKine, Upper Providence, Collegeville, PA, USA.

Abnormal accumulation of β-catenin protein, a key transcriptional activator required for Wnt signaling, is the hallmark of many tumor types, including colon cancer. In normal cells, β-catenin protein level is tightly controlled by a multiprotein complex through the proteosome pathway. Mutations in the components of the β-catenin degradation complex, such as adenomatous polyposis coli (APC) and Axin, lead to β-catenin stabilization and the constitutive activation of target genes. Since the signal transduction of Wnt/β-catenin is mainly mediated by protein-protein interactions, this pathway has been particularly refractory to conventional target-based small-molecule screening. Here we designed a cellular high-content imaging assay to detect β-catenin protein through immunofluorescent staining in the SW480 colon cancer cell line, which has elevated β-catenin endogenously. We demonstrate that the assay is robust and specific to screen a focused biologically diverse chemical library set against known targets that play diverse cellular functions. We identified a number of hits that reduce β-catenin levels without causing cell death. These hits may serve as tools to understand the dynamics of β-catenin degradation. This study demonstrates that detecting cell-based β-catenin protein stability is a viable approach to identifying novel mechanisms of β-catenin regulation as well as small molecules of therapeutic potential.
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http://dx.doi.org/10.1177/1087057115620169DOI Listing
March 2016

Cell-Based Selection Expands the Utility of DNA-Encoded Small-Molecule Library Technology to Cell Surface Drug Targets: Identification of Novel Antagonists of the NK3 Tachykinin Receptor.

ACS Comb Sci 2015 Dec 3;17(12):722-31. Epub 2015 Dec 3.

Molecular Discovery Research, GlaxoSmithKline , Waltham, Massachusetts 02451, United States.

DNA-encoded small-molecule library technology has recently emerged as a new paradigm for identifying ligands against drug targets. To date, this technology has been used with soluble protein targets that are produced and used in a purified state. Here, we describe a cell-based method for identifying small-molecule ligands from DNA-encoded libraries against integral membrane protein targets. We use this method to identify novel, potent, and specific inhibitors of NK3, a member of the tachykinin family of G-protein coupled receptors (GPCRs). The method is simple and broadly applicable to other GPCRs and integral membrane proteins. We have extended the application of DNA-encoded library technology to membrane-associated targets and demonstrate the feasibility of selecting DNA-tagged, small-molecule ligands from complex combinatorial libraries against targets in a heterogeneous milieu, such as the surface of a cell.
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http://dx.doi.org/10.1021/acscombsci.5b00124DOI Listing
December 2015

A novel approach applying a chemical biology strategy in phenotypic screening reveals pathway-selective regulators of histone 3 K27 tri-methylation.

Mol Biosyst 2014 Feb;10(2):251-7

Molecular Discovery Research, GlaxoSmithKline, Collegeville, PA, USA.

Epigenetic regulation by histone methylation is crucial for proper programming of the genome during development. Homeostasis of histone methylation is balanced by the activities of histone methyltransferases and demethylases. Although these methyltransferases and demethylases represent logical targets for potential drug discovery, the activities of methyltransferases and demethylases regulated in response to a complex biological stimulus are also important and not yet clear. To manipulate and study histone methylation in biological systems, we screened a Biologically Diverse Compound Set (BDCS) utilizing a phenotypic assay system that directly measures the Histone 3 K27 tri-methylation (H3K27me3) level in cells. The BDCS is a unique set of target-annotated chemical probes, containing a total of 5853 compounds targeting 736 unique proteins with multiple maximally selective compounds for each target. A number of targets, with multiple hits against each target, were identified in the screen. This gave us confidence that these targets and pathways may be relevant, and included the identification of non-methyltransferase/demethylase targets as potential upstream regulators of H3K27me3. Our study suggests that a systematically designed chemical probe library can serve as a powerful drug discovery tool when combined with phenotypic screening. Follow-up studies using these findings may reveal novel therapeutically useful pathways and targets of H3K27me3 regulation.
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http://dx.doi.org/10.1039/c3mb70413kDOI Listing
February 2014

Development of phenotypic screening assays for γ-globin induction using primary human bone marrow day 7 erythroid progenitor cells.

J Biomol Screen 2013 Dec 25;18(10):1212-22. Epub 2013 Oct 25.

1Molecular Discovery Research, GlaxoSmithKline, Collegeville, PA, USA.

Sickle cell anemia (SCA) is a genetic disorder of the β-globin gene. SCA results in chronic ischemia with pain and tissue injury. The extent of SCA symptoms can be ameliorated by treatment with drugs, which result in increasing the levels of γ-globin in patient red blood cells. Hydroxyurea (HU) is a Food and Drug Administration-approved drug for SCA, but it has dose-limiting toxicity, and patients exhibit highly variable treatment responses. To identify compounds that may lead to the development of better and safer medicines, we have established a method using primary human bone marrow day 7 erythroid progenitor cells (EPCs) to screen for compounds that induce γ-globin production. First, human marrow CD34(+) cells were cultured and expanded for 7 days and characterized for the expression of erythroid differentiation markers (CD71, CD36, and CD235a). Second, fresh or cryopreserved EPCs were treated with compounds for 3 days in 384-well plates followed by γ-globin quantification by an enzyme-linked immunosorbent assay (ELISA), which was validated using HU and decitabine. From the 7408 compounds screened, we identified at least one new compound with confirmed γ-globin-inducing activity. Hits are undergoing analysis in secondary assays. In this article, we describe the method of generating fit-for-purpose EPCs; the development, optimization, and validation of the ELISA and secondary assays for γ-globin detection; and screening results.
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http://dx.doi.org/10.1177/1087057113499776DOI Listing
December 2013

Development and validation of reagents and assays for EZH2 peptide and nucleosome high-throughput screens.

J Biomol Screen 2012 Dec 17;17(10):1279-92. Epub 2012 Aug 17.

Platform Technology and Science, GlaxoSmithKline, Collegeville, PA 19426, USA.

Histone methyltransferases (HMT) catalyze the methylation of histone tail lysines, resulting in changes in gene transcription. Misregulation of these enzymes has been associated with various forms of cancer, making this target class a potential new area for the development of novel chemotherapeutics. EZH2 is the catalytic component of the polycomb group repressive complex (PRC2), which selectively methylates histone H3 lysine 27 (H3K27). EZH2 is overexpressed in prostate, breast, bladder, brain, and other tumor types and is recognized as a molecular marker for cancer progression and aggressiveness. Several new reagents and assays were developed to aid in the identification of EZH2 inhibitors, and these were used to execute two high-throughput screening campaigns. Activity assays using either an H3K27 peptide or nucleosomes as substrates for methylation are described. The strategy to screen EZH2 with either a surrogate peptide or a natural substrate led to the identification of the same tractable series. Compounds from this series are reversible, are [(3)H]-S-adenosyl-L-methionine competitive, and display biochemical inhibition of H3K27 methylation.
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http://dx.doi.org/10.1177/1087057112453765DOI Listing
December 2012

Perspectives on the discovery of small-molecule modulators for epigenetic processes.

J Biomol Screen 2012 Jun 5;17(5):555-71. Epub 2012 Mar 5.

GlaxoSmithKline, Collegeville, Pennsylvania, USA.

Epigenetic gene regulation is a critical process controlling differentiation and development, the malfunction of which may underpin a variety of diseases. In this article, we review the current landscape of small-molecule epigenetic modulators including drugs on the market, key compounds in clinical trials, and chemical probes being used in epigenetic mechanistic studies. Hit identification strategies for the discovery of small-molecule epigenetic modulators are summarized with respect to writers, erasers, and readers of histone marks. Perspectives are provided on opportunities for new hit discovery approaches, some of which may define the next generation of therapeutic intervention strategies for epigenetic processes.
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http://dx.doi.org/10.1177/1087057112437763DOI Listing
June 2012

A standard operating procedure for assessing liquid handler performance in high-throughput screening.

J Biomol Screen 2002 Dec;7(6):554-69

GlaxoSmithKline Pharmaceuticals, Department of Molecular Screening, King of Prussia, PA, USA.

The thrust of early drug discovery in recent years has been toward the configuration of homogeneous miniaturized assays. This has allowed organizations to contain costs in the face of exponential increases in the number of screening assays that need to be run to remain competitive. Miniaturization brings with it an increasing dependence on instrumentation, which over the past several years has seen the development of nanodispensing capability and sophisticated detection strategies. To maintain confidence in the data generated from miniaturized assays, it is critical to ensure that both compounds and reagents have been delivered as expected to the target wells. The authors have developed a standard operating procedure for liquid-handling quality control that has enabled them to evaluate performance on 2 levels. The first level provides for routine daily testing on existing instrumentation, and the second allows for more rigorous testing of new dispensing technologies. The procedure has shown itself to be useful in identifying both method programming and instrumentation performance shortcomings and has provided a means to harmonizing instrumentation usage by assay development and screening groups. The goal is that this type of procedure be used for facilitating the exchange of liquid handler performance data across the industry.
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http://dx.doi.org/10.1177/1087057102238630DOI Listing
December 2002

Single-molecule detection technologies in miniaturized high-throughput screening: fluorescence intensity distribution analysis.

J Biomol Screen 2003 Feb;8(1):19-33

GlaxoSmithKline, Direvo, Cologne, Germany.

Single-molecule detection technologies are becoming a powerful readout format to support ultra-high-throughput screening. These methods are based on the analysis of fluorescence intensity fluctuations detected from a small confocal volume element. The fluctuating signal contains information about the mass and brightness of the different species in a mixture. The authors demonstrate a number of applications of fluorescence intensity distribution analysis (FIDA), which discriminates molecules by their specific brightness. Examples for assays based on brightness changes induced by quenching/dequenching of fluorescence, fluorescence energy transfer, and multiple-binding stoichiometry are given for important drug targets such as kinases and proteases. FIDA also provides a powerful method to extract correct biological data in the presence of compound fluorescence.
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http://dx.doi.org/10.1177/1087057102239669DOI Listing
February 2003

Conformational restriction of methionyl tRNA synthetase inhibitors leading to analogues with potent inhibition and excellent gram-positive antibacterial activity.

Bioorg Med Chem Lett 2003 Apr;13(7):1265-8

GlaxoSmithKline, New Frontiers Science Park, Third Avenue, Harlow, Essex, UK.

Conformationally restricted analogues of the central linker unit of bacterial methionyl tRNA synthetase (MRS) inhibitors have been prepared. The (1S,2R)-cyclopentylmethyl moiety was identified as the preferred cyclic linker, with significant diastereo- and enantioselectivity of activity. Combination of this linker with an optimal substituted aryl right-hand side has resulted in a compound with exceptionally good antibacterial activity against staphylococci and enterococci, including antibiotic resistant strains.
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http://dx.doi.org/10.1016/s0960-894x(03)00093-3DOI Listing
April 2003

The antimicrobial natural product chuangxinmycin and some synthetic analogues are potent and selective inhibitors of bacterial tryptophanyl tRNA synthetase.

Bioorg Med Chem Lett 2002 Nov;12(21):3171-4

GlaxoSmithKline, New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW, UK.

The antimicrobial natural product chuangxinmycin has been found to be a potent and selective inhibitor of bacterial tryptophanyl tRNA synthetase (WRS). A number of analogues have been synthesised. The interaction with WRS appears to be highly constrained, as only sterically smaller analogues afforded significant inhibition. The only analogue to show inhibition comparable to chuangxinmycin also had antibacterial activity. WRS inhibition may contribute to the antibacterial action of chuangxinmycin.
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http://dx.doi.org/10.1016/s0960-894x(02)00604-2DOI Listing
November 2002

Nanomolar inhibitors of Staphylococcus aureus methionyl tRNA synthetase with potent antibacterial activity against gram-positive pathogens.

J Med Chem 2002 May;45(10):1959-62

Potent nanomolar inhibitors of Staphylococcus aureus methionyl tRNA synthetase have been derived from a file compound high throughput screening hit. Optimized compounds show excellent antibacterial activity against staphylococcal and enterococcal pathogens, including strains resistant to clinical antibiotics. Compound 11 demonstrated in vivo efficacy in an S. aureus rat abscess infection model.
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http://dx.doi.org/10.1021/jm025502xDOI Listing
May 2002