Publications by authors named "Glenn Hofmann"

16 Publications

  • Page 1 of 1

Screen for modulators of atonal homolog 1 gene expression using notch pathway-relevant gene transcription based cellular assays.

PLoS One 2018 12;13(12):e0207140. Epub 2018 Dec 12.

R&D Alternative Discovery and Development, GlaxoSmithKline, Stevenage, Hertfordshire, United Kingdom.

Atonal homolog 1 (Atoh1) is a basic helix-loop-helix 9 (bHLH) transcription factor acting downstream of Notch and is required for the differentiation of sensory hair cells in the inner ear and the specification of secretory cells during the intestinal crypt cell regeneration. Motivated by the observations that the upregulation of Atoh1 gene expression, through genetic manipulation or pharmacological inhibition of Notch signaling (e.g. γ-secretase inhibitors, GSIs), induces ectopic hair cell growth in the cochlea of the inner ear and partially restores hearing after injuries in experimental models, we decided to identify small molecule modulators of the Notch-Atoh1 pathway, which could potentially regenerate hair cells. However, the lack of cellular models of the inner ear has precluded the screening and characterization of such modulators. Here we report using a colon cancer cell line LS-174T, which displays Notch inhibition-dependent Atoh1 expression as a surrogate cellular model to screen for inducers of Atoh1 expression. We designed an Atoh1 promoter-driven luciferase assay to screen a target-annotated library of ~6000 compounds. We further developed a medium throughput, real-time quantitative RT-PCR assay measuring the endogenous Atoh1 gene expression to confirm the hits and eliminate false positives from the reporter-based screen. This strategy allowed us to successfully recover GSIs of known chemotypes. This LS-174T cell-based assay directly measures Atoh1 gene expression induced through Notch-Hes1 inhibition, and therefore offers an opportunity to identify novel cellular modulators along the Notch-Atoh1 pathway.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0207140PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6291236PMC
April 2019

Development of a High-Throughput Cul3-Keap1 Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay for Identifying Nrf2 Activators.

SLAS Discov 2019 02 1;24(2):175-189. Epub 2018 Nov 1.

1 Screening, Profiling and Mechanistic Biology, Platform Technology and Science, GlaxoSmithKline, Collegeville, PA, USA.

Nrf2, a master regulator of the phase II gene response to stress, is kept at low concentrations in the cell through binding to Keap1, an adaptor protein for the Cul3 ubiquitin ligase complex. To identify Nrf2 activators, two separate time-resolved fluorescence resonance energy transfer (TR-FRET) assays were developed to monitor the binding of Nrf2-Keap1 and Cul3-Keap1, respectively. The triterpenoid, 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl] imidazole (CDDO-Im) and its analogs, exhibited approximately 100-fold better potency in the Cul3-Keap1 assay than in the Nrf2-Keap1 assay, and this difference was more profound at 37 °C than at room temperature in the Nrf2-Keap1 assay, but this phenomenon was not observed in the Cul3-Keap1 assay. A full diversity screen of approximately 2,200,000 GSK compounds was run with the Cul3-Keap1 TR-FRET assay and multiple chemical series were identified and characterized.
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http://dx.doi.org/10.1177/2472555218807698DOI Listing
February 2019

High throughput screening identifies ATP-competitive inhibitors of the NLRP1 inflammasome.

Bioorg Med Chem Lett 2015 Jul 19;25(14):2739-43. Epub 2015 May 19.

Pattern Recognition Receptor DPU, GlaxoSmithKline, Collegeville Road, Collegeville, PA 19426, USA.

Nod-like receptors (NLRs) are cytoplasmic pattern recognition receptors that are promising targets for the development of anti-inflammatory therapeutics. Drug discovery efforts targeting NLRs have been hampered by their inherent tendency to form aggregates making protein generation and the development of screening assays very challenging. Herein we report the results of an HTS screen of NLR family member NLRP1 (NLR family, pyrin domain-containing 1) which was achieved through the large scale generation of recombinant GST-His-Thrombin-NLRP1 protein. The screen led to the identification of a diverse set of ATP competitive inhibitors with micromolar potencies. Activity of these hits was confirmed in a FP binding assay, and two homology models were employed to predict the possible binding mode of the leading series and facilitate further lead-optimization. These results highlight a promising strategy for the identification of inhibitors of NLR family members which are rapidly emerging as key drivers of inflammation in human disease.
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http://dx.doi.org/10.1016/j.bmcl.2015.05.032DOI Listing
July 2015

Selective class IIa histone deacetylase inhibition via a nonchelating zinc-binding group.

Nat Chem Biol 2013 May 24;9(5):319-25. Epub 2013 Mar 24.

Tempero Pharmaceuticals, Cambridge, Massachusetts, USA.

In contrast to studies on class I histone deacetylase (HDAC) inhibitors, the elucidation of the molecular mechanisms and therapeutic potential of class IIa HDACs (HDAC4, HDAC5, HDAC7 and HDAC9) is impaired by the lack of potent and selective chemical probes. Here we report the discovery of inhibitors that fill this void with an unprecedented metal-binding group, trifluoromethyloxadiazole (TFMO), which circumvents the selectivity and pharmacologic liabilities of hydroxamates. We confirm direct metal binding of the TFMO through crystallographic approaches and use chemoproteomics to demonstrate the superior selectivity of the TFMO series relative to a hydroxamate-substituted analog. We further apply these tool compounds to reveal gene regulation dependent on the catalytic active site of class IIa HDACs. The discovery of these inhibitors challenges the design process for targeting metalloenzymes through a chelating metal-binding group and suggests therapeutic potential for class IIa HDAC enzyme blockers distinct in mechanism and application compared to current HDAC inhibitors.
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http://dx.doi.org/10.1038/nchembio.1223DOI Listing
May 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

Discovery and characterization of a cell-permeable, small-molecule c-Abl kinase activator that binds to the myristoyl binding site.

Chem Biol 2011 Feb;18(2):177-86

Oncology Research and Development, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, PA 19426, USA.

c-Abl kinase activity is regulated by a unique mechanism involving the formation of an autoinhibited conformation in which the N-terminal myristoyl group binds intramolecularly to the myristoyl binding site on the kinase domain and induces the bending of the αI helix that creates a docking surface for the SH2 domain. Here, we report a small-molecule c-Abl activator, DPH, that displays potent enzymatic and cellular activity in stimulating c-Abl activation. Structural analyses indicate that DPH binds to the myristoyl binding site and prevents the formation of the bent conformation of the αI helix through steric hindrance, a mode of action distinct from the previously identified allosteric c-Abl inhibitor, GNF-2, that also binds to the myristoyl binding site. DPH represents the first cell-permeable, small-molecule tool compound for c-Abl activation.
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http://dx.doi.org/10.1016/j.chembiol.2010.12.013DOI Listing
February 2011

Assay development and high-throughput screening of small molecular c-Abl kinase activators.

J Biomol Screen 2011 Jan 11;16(1):53-64. Epub 2010 Oct 11.

Biological Reagents and Assay Development, GlaxoSmithKline Pharmaceuticals, Collegeville, PA, USA.

A 2-step kinase assay was developed and used in a high-throughput screen (HTS) of more than 1 million compounds in an effort to identify c-Abl tyrosine kinase activators. This assay employed a 2-step phosphorylation reaction: in the first step, purified recombinant c-Abl was activated by incubating with compound in the presence of adenosine triphosphate (ATP). In the second step, the TAMRA-labeled IMAP Abltide substrate was added to allow phosphorylation of the substrate to occur. The assay was calibrated such that inactive c-Abl protein was activated by ATP alone to a degree that it not only demonstrated a measurable c-Abl activity but also maintained a robust assay window for screening. The screen resulted in 8624 primary hits with >30% response. Further analysis showed that 1024 had EC(50) <10 µM with a max % response of >50%. These hits were structurally and chemically diverse with possibly different mechanisms for activating c-Abl. In addition, selective hits were shown to be cell permeable and were able to induce c-Abl activation as determined by In-Cell Western (ICW) analysis of HEK-MSRII cells transduced with BacMam virus expressing full-length c-Abl.
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http://dx.doi.org/10.1177/1087057110384133DOI Listing
January 2011

Process validation and screen reproducibility in high-throughput screening.

J Biomol Screen 2009 Jan;14(1):66-76

GlaxoSmithKline R&D Pharmaceuticals, Screening and Compound Profiling, Tres Cantos, Spain.

The use of large-scale compound screening has become a key component of drug discovery projects in both the pharmaceutical and the biotechnological industries. More recently, these activities have also been embraced by the academic community as a major tool for chemical genomic activities. High-throughput screening (HTS) activities constitute a major step in the initial drug discovery efforts and involve the use of large quantities of biological reagents, hundreds of thousands to millions of compounds, and the utilization of expensive equipment. All these factors make it very important to evaluate in advance of the HTS campaign any potential issues related to reproducibility of the experimentation and the quality of the results obtained at the end of these very costly activities. In this article, the authors describe how GlaxoSmithKline (GSK) has addressed the need of a true validation of the HTS process before embarking in full HTS campaigns. They present 2 different aspects of the so-called validation process: (1) optimization of the HTS workflow and its validation as a quality process and (2) the statistical evaluation of the HTS, focusing on the reproducibility of results and the ability to distinguish active from nonactive compounds in a vast collection of samples. The authors describe a variety of reproducibility indexes that are either innovative or have been adapted from generic medical diagnostic screening strategies. In addition, they exemplify how these validation tools have been implemented in a number of case studies at GSK.
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http://dx.doi.org/10.1177/1087057108326664DOI Listing
January 2009

Highly potent inhibitors of methionine aminopeptidase-2 based on a 1,2,4-triazole pharmacophore.

J Med Chem 2007 Aug 18;50(16):3777-85. Epub 2007 Jul 18.

Department of Medicinal Chemistry, Enzymology, Oncology, and Structural Biology, GlaxoSmithkline, King of Prussia, PA 19406, USA.

High-throughput screening for inhibitors of the human metalloprotease, methionine aminopeptidase-2 (MetAP2), identified a potent class of 3-anilino-5-benzylthio-1,2,4-triazole compounds. Efficient array and interative synthesis of triazoles led to rapid SAR development around the aniline, benzylthio, and triazole moeities. Evaluation of these analogs in a human MetAP2 enzyme assay led to the identification of several inhibitors with potencies in the 50-100 picomolar range. The deleterious effects on inhibitor potency by methylation of the anilino-triazole nitrogens, as well as the X-ray crystal structure of triazole 102 bound in the active site of MetAP2, confirm the key interactions between the triazole nitrogens, the active site cobalt atoms, and the His-231 side-chain. The structure has also provided a rationale for interpreting SAR within the triazole series. Key aniline (2-isopropylphenyl) and sulfur substituents (furanylmethyl) identified in the SAR studies led to the identification of potent inhibitors (103 and 104) of endothelial cell proliferation. Triazoles 103 and 104 also exhibited dose-dependent activity in an aortic ring tissue model of angiogenesis highlighting the potential utility of MetAP2 inhibitors as anticancer agents.
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http://dx.doi.org/10.1021/jm061182wDOI Listing
August 2007

Studies on acyl pyrrolidine inhibitors of HCV RNA-dependent RNA polymerase to identify a molecule with replicon antiviral activity.

Bioorg Med Chem Lett 2007 Apr 19;17(7):1930-3. Epub 2007 Jan 19.

GlaxoSmithKline Pharmaceuticals, 1250 South Collegeville Road, PO Box 5089, Collegeville, PA 19426-0989, USA.

The SAR development is described for a series of N-acyl pyrrolidine inhibitors of the Hepatitis C virus RNA-dependent RNA polymerase, NS5B, from tractable Delta21 enzyme inhibitors to an example with antiviral activity in a cellular assay (HCV replicon).
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http://dx.doi.org/10.1016/j.bmcl.2007.01.034DOI Listing
April 2007

3-(1,1-dioxo-2H-(1,2,4)-benzothiadiazin-3-yl)-4-hydroxy-2(1H)-quinolinones, potent inhibitors of hepatitis C virus RNA-dependent RNA polymerase.

J Med Chem 2006 Feb;49(3):971-83

Department of Medicinal Chemistry and Drug Metabolism, the Musculoskeletal, Microbial and Proliferative Diseases Center of Excellence for Drug Discovery, GlaxoSmithKline Pharmaceuticals, Collegeville, PA 19426, USA.

Recently, we disclosed a new class of HCV polymerase inhibitors discovered through high-throughput screening (HTS) of the GlaxoSmithKline proprietary compound collection. This interesting class of 3-(1,1-dioxo-2H-1,2,4-benzothiadiazin-3-yl)-4-hydroxy-2(1H)-quinolinones potently inhibits HCV polymerase enzymatic activity and inhibits the ability of the subgenomic HCV replicon to replicate in Huh-7 cells. This report will focus on the structure-activity relationships (SAR) of substituents on the quinolinone ring, culminating in the discovery of 1-(2-cyclopropylethyl)-3-(1,1-dioxo-2H-1,2,4-benzothiadiazin-3-yl)-6-fluoro-4-hydroxy-2(1H)-quinolinone (130), an inhibitor with excellent potency in biochemical and cellular assays possessing attractive molecular properties for advancement as a clinical candidate. The potential for development and safety assessment profile of compound 130 will also be discussed.
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http://dx.doi.org/10.1021/jm050855sDOI Listing
February 2006

4-Aryl-1,2,3-triazole: a novel template for a reversible methionine aminopeptidase 2 inhibitor, optimized to inhibit angiogenesis in vivo.

J Med Chem 2005 Sep;48(18):5644-7

GlaxoSmithKline Pharmaceuticals, King of Prussia, Pennsylvania 19406, USA.

Inhibitors of human methionine aminopeptidase type 2 (hMetAP2) are of interest as potential treatments for cancer. A new class of small molecule reversible inhibitors of hMetAP2 was discovered and optimized, the 4-aryl-1,2,3-triazoles. Compound 24, a potent inhibitor of cobalt-activated hMetAP2, also inhibits human and mouse endothelial cell growth. Using a mouse matrigel model, this reversible hMetAP2 inhibitor was also shown to inhibit angiogenesis in vivo.
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http://dx.doi.org/10.1021/jm050408cDOI Listing
September 2005

Identification of small molecule inhibitors of the hepatitis C virus RNA-dependent RNA polymerase from a pyrrolidine combinatorial mixture.

Bioorg Med Chem Lett 2005 Mar;15(6):1553-6

GlaxoSmithKline Pharmaceuticals, 1250 South Collegeville Road, PO Box 5089, Collegeville, PA 19426-0989, USA.

HTS of the compound collection for inhibition of the HCV RNA dependent RNA polymerase identified two 168 member N-acyl pyrrolidine combinatorial mixture hits. Deconvolution and expansion of these mixtures by solid phase synthesis to establish initial SAR and identify a potent inhibitor is reported.
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http://dx.doi.org/10.1016/j.bmcl.2005.01.076DOI Listing
March 2005

Investigations on anthocyanins in wines from Vitis vinifera cv. pinotage: factors influencing the formation of pinotin A and its correlation with wine age.

J Agric Food Chem 2004 Feb;52(3):498-504

Institute of Food Chemistry, Technical University of Braunschweig, Schleinitzstrasse 20, 38106 Braunschweig, Germany.

Pinotage red wines were found to contain a reaction product of malvidin 3-glucoside and caffeic acid, the so-called pinotin A. A total of 50 Pinotage wines from the vintages 1996-2002 were analyzed for the content of pinotin A, malvidin 3-glucoside, caffeic acid, and caftaric acid. Statistical analyses were performed to reveal variations in the content of these compounds and to determine the factors that influence pinotin A formation during wine aging. An exponential increase of the concentration of this aging product was observed with prolonged storage time. The most rapid synthesis of pinotin A was observed in 2.5-4 year old wines, although at this age malvidin 3-glucoside is already degraded to a large extent. This phenomenon is explained by the increased ratio of caffeic acid/malvidin 3-glucoside, which strongly favors the formation of pinotin A and makes side reactions less likely. Pinotin A formation proceeds as long as a certain level of malvidin 3-glucoside is maintained in the wines. In wines >5-6 years old degradation or polymerization of pinotin A finally exceeds the rate of its de novo synthesis.
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http://dx.doi.org/10.1021/jf035034fDOI Listing
February 2004

Identification of novel isoform-selective inhibitors within class I histone deacetylases.

J Pharmacol Exp Ther 2003 Nov 15;307(2):720-8. Epub 2003 Sep 15.

Department of Vascular Biology, GlaxoSmithKline, Rm. 25-1084, 709 Swedeland Road, King of Prussia, PA 19406, USA.

Histone deacetylases (HDACs) represent an expanding family of protein modifying-enzymes that play important roles in cell proliferation, chromosome remodeling, and gene transcription. We have previously shown that recombinant human HDAC8 can be expressed in bacteria and retain its catalytic activity. To further explore the catalytic activity of HDACs, we expressed two additional human class I HDACs, HDAC1 and HDAC3, in baculovirus. Recombinant HDAC1 and HDAC3 fusion proteins remained soluble and catalytically active and were purified to near homogeneity. Interestingly, trichostatin (TSA) was found to be a potent inhibitor for all three HDACs (IC50 value of approximately 0.1-0.3 microM), whereas another HDAC inhibitor MS-27-275 (N-(2-aminophenyl)-4-[N-(pyridin-3-methyloxycarbonyl)-aminomethyl]benzamide) preferentially inhibited HDAC1 (IC50 value of approximately 0.3 microM) versus HDAC3 (IC50 value of approximately 8 microM) and had no inhibitory activity toward HDAC8 (IC50 value >100 microM). MS-27-275 as well as TSA increased histone H4 acetylation, induced apoptosis in the human colon cancer cell line SW620, and activated the simian virus 40 early promoter. HDAC1 protein was more abundantly expressed in SW620 cells compared with that of HDAC3 and HDAC8. Using purified recombinant HDAC proteins, we identified several novel HDAC inhibitors that preferentially inhibit HDAC1 or HDAC8. These inhibitors displayed distinct properties in inducing histone acetylation and reporter gene expression. These results suggest selective HDAC inhibitors could be identified using recombinantly expressed HDACs and that HDAC1 may be a promising therapeutic target for designing HDAC inhibitors for proliferative diseases such as cancer.
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http://dx.doi.org/10.1124/jpet.103.055541DOI Listing
November 2003

Identification and biological characterization of heterocyclic inhibitors of the hepatitis C virus RNA-dependent RNA polymerase.

J Biol Chem 2002 Oct 6;277(41):38322-7. Epub 2002 Aug 6.

Department of Medicinal Chemistry, The Musculoskeletal, Microbial and Proliferative Diseases Center of Excellence for Drug Discovery, GlaxoSmithKline Pharmaceuticals, Collegeville, Pennsylvania 19426, USA.

The hepatitis C virus (HCV) NS5B protein encodes an RNA-dependent RNA polymerase (RdRp), the primary catalytic enzyme of the HCV replicase complex. We established a biochemical RNA synthesis assay, using purified recombinant NS5B lacking the C-terminal 21 amino acid residues, to identify potential polymerase inhibitors from a high throughput screen of the GlaxoSmithKline proprietary compound collection. The benzo-1,2,4-thiadiazine compound 1 was found to be a potent, highly specific inhibitor of NS5B. This agent interacts directly with the viral polymerase and inhibits RNA synthesis in a manner noncompetitive with respect to GTP. Furthermore, in the absence of an in vitro-reconstituted HCV replicase assay employing viral and host proteins, the ability of compound 1 to inhibit NS5B-directed viral RNA replication was determined using the Huh7 cell-based HCV replicon system. Compound 1 reduced viral RNA in replicon cells with an IC(50) of approximately 0.5 microm, suggesting that the inhibitor was able to access the perinuclear membrane and inhibit the polymerase activity in the context of a replicase complex. Preliminary structure-activity studies on compound 1 led to the identification of a modified inhibitor, compound 4, showing an improvement in both biochemical and cell-based potency. Lastly, data are presented suggesting that these compounds interfere with the formation of negative and positive strand progeny RNA by a similar mode of action. Investigations are ongoing to assess the potential utility of such agents in the treatment of chronic HCV disease.
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http://dx.doi.org/10.1074/jbc.M205566200DOI Listing
October 2002