Publications by authors named "Eugene L Stewart"

30 Publications

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Complementary NAD replacement strategies fail to functionally protect dystrophin-deficient muscle.

Skelet Muscle 2020 10 22;10(1):30. Epub 2020 Oct 22.

Muscle Metabolism Unit, GlaxoSmithKline R&D, Research Triangle Park, NC, Collegeville, PA, USA.

Background: Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disorder stemming from a loss of functional dystrophin. Current therapeutic options for DMD are limited, as small molecule modalities remain largely unable to decrease the incidence or mitigate the consequences of repetitive mechanical insults to the muscle during eccentric contractions (ECCs).

Methods: Using a metabolomics-based approach, we observed distinct and transient molecular phenotypes in muscles of dystrophin-deficient MDX mice subjected to ECCs. Among the most chronically depleted metabolites was nicotinamide adenine dinucleotide (NAD), an essential metabolic cofactor suggested to protect muscle from structural and metabolic degeneration over time. We tested whether the MDX muscle NAD pool can be expanded for therapeutic benefit using two complementary small molecule strategies: provision of a biosynthetic precursor, nicotinamide riboside, or specific inhibition of the NAD-degrading ADP-ribosyl cyclase, CD38.

Results: Administering a novel, potent, and orally available CD38 antagonist to MDX mice successfully reverted a majority of the muscle metabolome toward the wildtype state, with a pronounced impact on intermediates of the pentose phosphate pathway, while supplementing nicotinamide riboside did not significantly affect the molecular phenotype of the muscle. However, neither strategy sustainably increased the bulk tissue NAD pool, lessened muscle damage markers, nor improved maximal hindlimb strength following repeated rounds of eccentric challenge and recovery.

Conclusions: In the absence of dystrophin, eccentric injury contributes to chronic intramuscular NAD depletion with broad pleiotropic effects on the molecular phenotype of the tissue. These molecular consequences can be more effectively overcome by inhibiting the enzymatic activity of CD38 than by supplementing nicotinamide riboside. However, we found no evidence that either small molecule strategy is sufficient to restore muscle contractile function or confer protection from eccentric injury, undermining the modulation of NAD metabolism as a therapeutic approach for DMD.
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http://dx.doi.org/10.1186/s13395-020-00249-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7579925PMC
October 2020

The exploration of aza-quinolines as hematopoietic prostaglandin D synthase (H-PGDS) inhibitors with low brain exposure.

Bioorg Med Chem 2020 12 3;28(23):115791. Epub 2020 Oct 3.

GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, PA 19426, USA.

GlaxoSmithKline and Astex Pharmaceuticals recently disclosed the discovery of the potent H-PGDS inhibitor GSK2894631A 1a (IC = 9.9 nM) as part of a fragment-based drug discovery collaboration with Astex Pharmaceuticals. This molecule exhibited good murine pharmacokinetics, allowing it to be utilized to explore H-PGDS pharmacology in vivo. Yet, with prolonged dosing at higher concentrations, 1a induced CNS toxicity. Looking to attenuate brain penetration in this series, aza-quinolines, were prepared with the intent of increasing polar surface area. Nitrogen substitutions at the 6- and 8-positions of the quinoline were discovered to be tolerated by the enzyme. Subsequent structure activity studies in these aza-quinoline scaffolds led to the identification of 1,8-naphthyridine 1y (IC = 9.4 nM) as a potent peripherally restricted H-PGDS inhibitor. Compound 1y is efficacious in four in vivo inflammatory models and exhibits no CNS toxicity.
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http://dx.doi.org/10.1016/j.bmc.2020.115791DOI Listing
December 2020

The discovery of quinoline-3-carboxamides as hematopoietic prostaglandin D synthase (H-PGDS) inhibitors.

Bioorg Med Chem 2019 04 11;27(8):1456-1478. Epub 2019 Feb 11.

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

With the goal of discovering more selective anti-inflammatory drugs, than COX inhibitors, to attenuate prostaglandin signaling, a fragment-based screen of hematopoietic prostaglandin D synthase was performed. The 76 crystallographic hits were sorted into similar groups, with the 3-cyano-quinoline 1a (FP IC = 220,000 nM, LE = 0.43) being a potent member of the 6,6-fused heterocyclic cluster. Employing SAR insights gained from structural comparisons of other H-PGDS fragment binding mode clusters, the initial hit 1a was converted into the 70-fold more potent quinoline 1d (IC = 3,100 nM, LE = 0.49). A systematic substitution of the amine moiety of 1d, utilizing structural information and array chemistry, with modifications to improve inhibitor stability, resulted in the identification of the 300-fold more active H-PGDS inhibitor tool compound 1bv (IC = 9.9 nM, LE = 0.42). This selective inhibitor exhibited good murine pharmacokinetics, dose-dependently attenuated PGD production in a mast cell degranulation assay and should be suitable to further explore H-PGDS biology.
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http://dx.doi.org/10.1016/j.bmc.2019.02.017DOI Listing
April 2019

2,4-Diamino-8-quinazoline carboxamides as novel, potent inhibitors of the NAD hydrolyzing enzyme CD38: Exploration of the 2-position structure-activity relationships.

Bioorg Med Chem 2018 05 15;26(8):2107-2150. Epub 2018 Mar 15.

GlaxoSmithKline Research and Development, 5 Moore Drive, P.O. Box 13398, Research Triangle Park, NC 27709, USA.

Starting from 4-amino-8-quinoline carboxamide lead 1a and scaffold hopping to the chemically more tractable quinazoline, a systematic exploration of the 2-substituents of the quinazoline ring, utilizing structure activity relationships and conformational constraint, resulted in the identification of 39 novel CD38 inhibitors. Eight of these analogs were 10-100-fold more potent human CD38 inhibitors, including the single digit nanomolar inhibitor 1am. Several of these molecules also exhibited improved therapeutic indices relative to hERG activity. A representative analog 1r exhibited suitable pharmacokinetic parameters for in vivo animal studies, including moderate clearance and good oral bioavailability. These inhibitor compounds will aid in the exploration of the enzymatic functions of CD38, as well as furthering the study of the therapeutic implications of NAD enhancement in metabolic disease models.
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http://dx.doi.org/10.1016/j.bmc.2018.03.021DOI Listing
May 2018

Discovery of 4-Amino-8-quinoline Carboxamides as Novel, Submicromolar Inhibitors of NAD-Hydrolyzing Enzyme CD38.

J Med Chem 2015 Sep 24;58(17):7021-56. Epub 2015 Aug 24.

GlaxoSmithKline Research and Development , 5 Moore Drive, P.O. Box 13398, Research Triangle Park, North Carolina 27709, United States.

Starting from the micromolar 8-quinoline carboxamide high-throughput screening hit 1a, a systematic exploration of the structure-activity relationships (SAR) of the 4-, 6-, and 8-substituents of the quinoline ring resulted in the identification of approximately 10-100-fold more potent human CD38 inhibitors. Several of these molecules also exhibited pharmacokinetic parameters suitable for in vivo animal studies, including low clearances and decent oral bioavailability. Two of these CD38 inhibitors, 1ah and 1ai, were shown to elevate NAD tissue levels in liver and muscle in a diet-induced obese (DIO) C57BL/6 mouse model. These inhibitor tool compounds will enable further biological studies of the CD38 enzyme as well as the investigation of the therapeutic implications of NAD enhancement in disease models of abnormally low NAD.
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http://dx.doi.org/10.1021/acs.jmedchem.5b00992DOI Listing
September 2015

Discovery, Synthesis, and Biological Evaluation of Thiazoloquin(az)olin(on)es as Potent CD38 Inhibitors.

J Med Chem 2015 Apr 10;58(8):3548-71. Epub 2015 Apr 10.

Research and Development, GlaxoSmithKline, 5 Moore Drive, P.O. Box 13398, Research Triangle Park, North Carolina 27709, United States.

A series of thiazoloquin(az)olinones were synthesized and found to have potent inhibitory activity against CD38. Several of these compounds were also shown to have good pharmacokinetic properties and demonstrated the ability to elevate NAD levels in plasma, liver, and muscle tissue. In particular, compound 78c was given to diet induced obese (DIO) C57Bl6 mice, elevating NAD > 5-fold in liver and >1.2-fold in muscle versus control animals at a 2 h time point. The compounds described herein possess the most potent CD38 inhibitory activity of any small molecules described in the literature to date. The inhibitors should allow for a more detailed assessment of how NAD elevation via CD38 inhibition affects physiology in NAD deficient states.
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http://dx.doi.org/10.1021/jm502009hDOI Listing
April 2015

A pre-steady state and steady state kinetic analysis of the N-ribosyl hydrolase activity of hCD157.

Arch Biochem Biophys 2014 Dec 20;564:156-63. Epub 2014 Sep 20.

Platform Technology and Science, Molecular Discovery Research, Chemical Sciences, GlaxoSmithKline, 5 Moore Drive, 3.2094, Research Triangle Park, NC 27709, United States.

hCD157 catalyzes the hydrolysis of nicotinamide riboside (NR) and nicotinic acid riboside (NAR). The release of nicotinamide or nicotinic acid from NR or NAR was confirmed by spectrophotometric, HPLC and NMR analyses. hCD157 is inactivated by a mechanism-based inhibitor, 2'-deoxy-2'-fluoro-nicotinamide arabinoside (fNR). Modification of the enzyme during the catalytic cycle by NR, NAR, or fNR increased the intrinsic protein fluorescence by approximately 50%. Pre-steady state and steady state data were used to derive a minimal kinetic scheme for the hydrolysis of NR. After initial complex formation a reversible step (360 and 30s(-1)) is followed by a slow irreversible step (0.1s(-1)) that defined the rate limiting step, or kcat. The calculated KMapp value for NR in the hydrolytic reaction is 6nM. The values of the kinetic constants suggest that one biological function of cell-surface hCD157 is to bind and slowly hydrolyze NR, possibly converting it to a ligand-activated receptor. Differences in substrate preference between hCD157 and hCD38 were rationalized through a comparison of the crystal structures of the two proteins. This comparison identified several residues in hCD157 (F108 and F173) that can potentially hinder the binding of dinucleotide substrates (NAD+).
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http://dx.doi.org/10.1016/j.abb.2014.09.008DOI Listing
December 2014

Discovery of a potent boronic acid derived inhibitor of the HCV RNA-dependent RNA polymerase.

J Med Chem 2014 Mar 29;57(5):1902-13. Epub 2013 May 29.

GlaxoSmithKline, Infectious Diseases Medicines Discovery Unit, 5 Moore Drive, Research Triangle Park, North Carolina 27709-3398, United States.

A boronic acid moiety was found to be a critical pharmacophore for enhanced in vitro potency against wild-type hepatitis C replicons and known clinical polymorphic and resistant HCV mutant replicons. The synthesis, optimization, and structure-activity relationships associated with inhibition of HCV replication in a subgenomic replication system for a series of non-nucleoside boron-containing HCV RNA-dependent RNA polymerase (NS5B) inhibitors are described. A summary of the discovery of 3 (GSK5852), a molecule which entered clinical trials in subjects infected with HCV in 2011, is included.
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http://dx.doi.org/10.1021/jm400317wDOI Listing
March 2014

Rational design of potent non-nucleoside inhibitors of HIV-1 reverse transcriptase.

J Med Chem 2012 Dec 26;55(23):10601-9. Epub 2012 Nov 26.

GlaxoSmithKline Research & Development, 5 Moore Drive, Research Triangle Park, North Carolina 27709, United States.

A new series of non-nucleoside reverse transcriptase inhibitors based on an imidazole-amide biarylether scaffold has been identified and shown to possess potent antiviral activity against HIV-1, including the NNRTI-resistant Y188L mutated virus. X-ray crystallography of inhibitors bound to reverse transcriptase, including a structure of the Y188L RT protein, was used extensively to help identify and optimize the key hydrogen-bonding motif. This led directly to the design of compound 43 that exhibits remarkable antiviral activity (EC50<1 nM) against a wide range of NNRTI-resistant viruses and a favorable pharmacokinetic profile across multiple species.
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http://dx.doi.org/10.1021/jm301294gDOI Listing
December 2012

Structure guided design of 5-arylindazole glucocorticoid receptor agonists and antagonists.

J Med Chem 2010 Jun;53(11):4531-44

Molecular Discovery Research, GlaxoSmithKline, Research Triangle Park, North Carolina 27709-3398, USA.

Glucocorticoid receptor (GR) agonists have been used for more than half a century as the most effective treatment of acute and chronic inflammatory conditions despite serious side effects that accompany their extended use that include glucose intolerance, muscle wasting, skin thinning, and osteoporosis. As a starting point for the identification of GR ligands with an improved therapeutic index, we wished to discover selective nonsteroidal GR agonists and antagonists with simplified structure compared to known GR ligands to serve as starting points for the optimization of dissociated GR modulators. To do so, we selected multiple chemical series by structure guided docking studies and evaluated GR agonist activity. From these efforts we identified 5-arylindazole compounds that showed moderate binding to the glucocorticoid receptor (GR) with clear opportunities for further development. Structure guided optimization was used to design arrays that led to potent GR agonists and antagonists. Several in vitro and in vivo experiments were utilized to demonstrate that GR agonist 23a (GSK9027) had a profile similar to that of a classical steroidal GR agonist.
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http://dx.doi.org/10.1021/jm100447cDOI Listing
June 2010

Discovery of tertiary sulfonamides as potent liver X receptor antagonists.

J Med Chem 2010 Apr;53(8):3412-6

GlaxoSmithKline, Five Moore Drive, Research Triangle Park, North Carolina 27709, USA.

Tertiary sulfonamides were identified in a HTS as dual liver X receptor (LXR, NR1H2, and NR1H3) ligands, and the binding affinity of the series was increased through iterative analogue synthesis. A ligand-bound cocrystal structure was determined which elucidated key interactions for high binding affinity. Further characterization of the tertiary sulfonamide series led to the identification of high affinity LXR antagonists. GSK2033 (17) is the first potent cell-active LXR antagonist described to date. 17 may be a useful chemical probe to explore the cell biology of this orphan nuclear receptor.
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http://dx.doi.org/10.1021/jm901797pDOI Listing
April 2010

Improving the developability profile of pyrrolidine progesterone receptor partial agonists.

Bioorg Med Chem Lett 2010 Jan 25;20(1):371-4. Epub 2009 Oct 25.

Department of Chemistry, Metabolic Pathways Centre for Excellence in Drug Discovery, GlaxoSmithKline Pharmaceuticals, 709 Swedeland Road, King of Prussia, PA 19406, USA.

The previously reported pyrrolidine class of progesterone receptor partial agonists demonstrated excellent potency but suffered from serious liabilities including hERG blockade and high volume of distribution in the rat. The basic pyrrolidine amine was intentionally converted to a sulfonamide, carbamate, or amide to address these liabilities. The evaluation of the degree of partial agonism for these non-basic pyrrolidine derivatives and demonstration of their efficacy in an in vivo model of endometriosis is disclosed herein.
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http://dx.doi.org/10.1016/j.bmcl.2009.10.092DOI Listing
January 2010

Discovery of orally active, pyrrolidinone-based progesterone receptor partial agonists.

Bioorg Med Chem Lett 2009 Aug 25;19(16):4664-8. Epub 2009 Jun 25.

Department of Chemistry, Metabolic Pathways Centre for Excellence in Drug Discovery, GlaxoSmithKline Pharmaceuticals, King of Prussia, PA 19406, USA.

We have designed and synthesized a novel series of pyrrolidinones as progesterone receptor partial agonists. Compounds from this series had improved AR selectivity, rat pharmacokinetic properties, and in vivo potency compared to the lead compound. In addition, these compounds had improved selectivity against hERG channel inhibition.
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http://dx.doi.org/10.1016/j.bmcl.2009.06.081DOI Listing
August 2009

Kinase-targeted library design through the application of the PharmPrint methodology.

J Chem Inf Model 2008 Dec;48(12):2395-403

GlaxoSmithKline, Five Moore Drive, Research Triangle Park, North Carolina 27709, USA.

The PharmPrint methodology, as modified and implemented by Deanda and Stewart, was prospectively evaluated for use as a virtual high-throughput screening tool by applying it to the design of target-focused arrays. To this end, PharmPrint quantitative structure-activity relationship (QSAR) models for the prediction of AKT1, Aurora-A, and ROCK1 inhibition were constructed and used to virtually screen two large combinatorial libraries. Based on predicted activities, an Aurora-A targeted array and a ROCK1 targeted array were designed and synthesized. One control group per designed array was also synthesized to assess the enrichment levels achieved by the QSAR models. For the Aurora-A targeted array, the hit rate, against the intended target, was 42.9%, whereas that of the control group was 0%. Thus, the enrichment level achieved by the Aurora-A QSAR model was incalculable. For the ROCK1 targeted array, the hit rate against the intended target was 30.6%, whereas that of the control group was 5.10%, making the enrichment level achieved by the ROCK1 QSAR model 6-fold above control. Clearly, these results support the use of the PharmPrint methodology as a virtual screening tool for the design of kinase-targeted arrays.
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http://dx.doi.org/10.1021/ci800276tDOI Listing
December 2008

The first X-ray crystal structure of the glucocorticoid receptor bound to a non-steroidal agonist.

Bioorg Med Chem Lett 2008 Dec 8;18(23):6097-9. Epub 2008 Oct 8.

Department of Computational and Structural Chemistry, Molecular Discovery Research, GlaxoSmithKline, 5 Moore Drive, Research Triangle Park, NC 27709, USA.

The amino-pyrazole 2,6-dichloro-N-ethyl benzamide 1 is a selective GR agonist with dexamethasone-like in vitro potency. Its X-ray crystal structure in the GR LBD (Glucocorticoid ligand-binding domain) is described and compared to other reported structures of steroidal GR agonists in the GR LBD (3E7C).
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http://dx.doi.org/10.1016/j.bmcl.2008.10.021DOI Listing
December 2008

X-ray crystal structure of the novel enhanced-affinity glucocorticoid agonist fluticasone furoate in the glucocorticoid receptor-ligand binding domain.

J Med Chem 2008 Jun 4;51(12):3349-52. Epub 2008 Jun 4.

An X-ray crystal structure is reported for the novel enhanced-affinity glucocorticoid agonist fluticasone furoate (FF) in the ligand binding domain of the glucocorticoid receptor. Comparison of this structure with those of dexamethasone and fluticasone propionate shows the 17 alpha furoate ester to occupy more fully the lipophilic 17 alpha pocket on the receptor, which may account for the enhanced glucocorticoid receptor binding of FF.
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http://dx.doi.org/10.1021/jm800279tDOI Listing
June 2008

A structural and in vitro characterization of asoprisnil: a selective progesterone receptor modulator.

Mol Endocrinol 2007 May 13;21(5):1066-81. Epub 2007 Mar 13.

Department of Computational, Analytical and Structural Sciences, GlaxoSmithKline Discovery Research, Research Triangle Park, North Carolina 27709, USA.

Selective progesterone receptor modulators (SPRMs) have been suggested as therapeutic agents for treatment of gynecological disorders. One such SPRM, asoprisnil, was recently in clinical trials for treatment of uterine fibroids and endometriosis. We present the crystal structures of progesterone receptor (PR) ligand binding domain complexed with asoprisnil and the corepressors nuclear receptor corepressor (NCoR) and SMRT. This is the first report of steroid nuclear receptor crystal structures with ligand and corepressors. These structures show PR in a different conformation than PR complexed with progesterone (P4). We profiled asoprisnil in PR-dependent assays to understand further the PR-mediated mechanism of action. We confirmed previous findings that asoprisnil demonstrated antagonism, but not agonism, in a PR-B transfection assay and the T47D breast cancer cell alkaline phosphatase activity assay. Asoprisnil, but not RU486, weakly recruited the coactivators SRC-1 and AIB1. However, asoprisnil strongly recruited the corepressor NCoR in a manner similar to RU486. Unlike RU486, NCoR binding to asoprisnil-bound PR could be displaced with equal affinity by NCoR or TIF2 peptides. We further showed that it weakly activated T47D cell gene expression of Sgk-1 and PPL and antagonized P4-induced expression of both genes. In rat leiomyoma ELT3 cells, asoprisnil demonstrated partial P4-like inhibition of cyclooxygenase (COX) enzymatic activity and COX-2 gene expression. In the rat uterotrophic assay, asoprisnil demonstrated no P4-like ability to oppose estrogen. Our data suggest that asoprisnil differentially recruits coactivators and corepressors compared to RU486 or P4, and this specific cofactor interaction profile is apparently insufficient to oppose estrogenic activity in rat uterus.
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http://dx.doi.org/10.1210/me.2006-0524DOI Listing
May 2007

Design and synthesis of an array of selective androgen receptor modulators.

J Comb Chem 2007 Jan-Feb;9(1):107-14

GlaxoSmithKline, Research Triangle Park, North Carolina 27709, USA.

We describe the design, using shape comparison and fast docking computer algorithms, and rapid parallel synthesis of a 1300 member array based on GSK7721, a 4-aminobenzonitrile androgen receptor (AR) antagonist identified by focused screening of the GSK compound collection. The array yielded 352 submicromolar and 17 subnanomolar AR agonists as measured by a cell-based reporter gene functional assay. The rapid synthesis of a large number of active compounds provided valuable information in the optimization of AR modulators, which may be useful in treating androgen deficiency in aging males.
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http://dx.doi.org/10.1021/cc060096eDOI Listing
March 2007

The evolution of progesterone receptor ligands.

Med Res Rev 2007 May;27(3):374-400

GlaxoSmithKline Inc., Research Triangle Park, North Carolina 27709, USA.

Progesterone is one of the first nuclear receptor hormones to be described functionally and subsequently approached as a drug target. Because progesterone (1) affects both menstruation and gestation via the progesterone receptor (PR), research aimed at modulating its activity is usually surrounded by controversy. However, ligands for PR were developed into drugs, and their evolution can be crudely divided into three periods: (1) drug-like steroids that mimic the gestational properties of progesterone; (2) drug-like steroids with different properties from progesterone and expanded therapeutic applications; and (3) non-steroidal PR ligands with improved selectivity and modulator properties and further expanded therapeutic applications. Although the latter have yet to see widespread clinical applications, their development is founded on a half century of research, and they represent the future for this drug target.
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http://dx.doi.org/10.1002/med.20083DOI Listing
May 2007

Array synthesis of progesterone receptor antagonists: 3-aryl-1,2-diazepines.

Bioorg Med Chem Lett 2006 Jul 5;16(14):3777-9. Epub 2006 May 5.

Discovery Research, GlaxoSmithKline, Research Triangle Park, NC 27709-3398, USA.

New non-steroidal chemotypes are required for the development of drugs targeting the steroid hormone receptors. The parallel array synthesis of 3-aryl-1,2-diazepines employing solid-supported reagents is described. The resulting compounds demonstrated high affinity binding to the progesterone receptor.
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http://dx.doi.org/10.1016/j.bmcl.2006.04.055DOI Listing
July 2006

A ligand-mediated hydrogen bond network required for the activation of the mineralocorticoid receptor.

J Biol Chem 2005 Sep 20;280(35):31283-93. Epub 2005 Jun 20.

Department of Gene Expression and Protein Biochemistry, GlaxoSmithKline, Research Triangle Park, North Carolina 27709, USA.

Ligand binding is the first step in hormone regulation of mineralocorticoid receptor (MR) activity. Here, we report multiple crystal structures of MR (NR3C2) bound to both agonist and antagonists. These structures combined with mutagenesis studies reveal that maximal receptor activation involves an intricate ligand-mediated hydrogen bond network with Asn770 which serves dual roles: stabilization of the loop preceding the C-terminal activation function-2 helix and direct contact with the hormone ligand. In addition, most activating ligands hydrogen bond to Thr945 on helix 10. Structural characterization of the naturally occurring S810L mutant explains how stabilization of a helix 3/helix 5 interaction can circumvent the requirement for this hydrogen bond network. Taken together, these results explain the potency of MR activation by aldosterone, the weak activation induced by progesterone and the antihypertensive agent spironolactone, and the binding selectivity of cortisol over cortisone.
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http://dx.doi.org/10.1074/jbc.M504098200DOI Listing
September 2005

Discovery of non-steroidal mifepristone mimetics: pyrazoline-based PR antagonists.

Bioorg Med Chem Lett 2005 Jul;15(13):3203-6

GlaxoSmithKline, Research Triangle Park, NC 27709-3398, USA.

Mifepristone is a non-selective antagonist of 3-oxosteroid receptors with both abortifacient and anti-endometriotic activities. Non-steroidal mimetics of mifepristone and progesterone are important templates for modulation of the progesterone receptor (PR). For our PR program, we sought an unexplored, synthetically accessible non-steroidal mimetic of mifepristone, suitable for parallel synthesis of analogues. Docking of compounds into a PR homology model identified 4-substituted pyrazolines, which, when synthesized and tested, exhibited functional antagonism of PR.
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http://dx.doi.org/10.1016/j.bmcl.2005.05.001DOI Listing
July 2005

Ab Initio and DFT Conformational Studies of Propanal, 2-Butanone, and Analogous Imines and Enamines.

J Chem Theory Comput 2005 Mar;1(2):230-8

Center for Drug Design, Department of Chemistry and Biochemistry, University of North Carolina at Greensboro, Greensboro, North Carolina 27402, Computational Center for Molecular Structure and Design, Department of Chemistry, University of Georgia, Athens, Georgia 30602, and Laboratory for Molecular Modeling, Division of Medicinal Chemistry and Natural Products, School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599.

The potential energy surfaces (PES) of 2-butanone, 2-butanimine, 1-butenamine, propanal, and propanimine have been explored with ab initio and DFT calculations at the RHF/6-311G**, MP2/6-311G**, and B3LYP/6-311G** levels of theory. In agreement with previous experimental and computational results, the PES provides two minima for each of the above molecules with the exception of 2-butanone, which clearly shows three distinct minima. Factors influencing the conformational preferences are also elaborated. Our calculations suggest that for 2-butanone and propanal, the steric and the bond dipole interactions are primarily responsible for the conformational preferences of these compounds. Additional charge-charge interactions might also play an important role in determining the imine conformations. For enamines, however, steric interactions play a critical role, with bond dipole interactions exerting some influence. Our results also suggest that for imine formation from butanone and/or propanal, the imine is the predominant product, not the enamine, which is consistent with experimental observations. Therefore, these calculations should provide a better understanding of the ketone/aldehyde to imine and enamine transformations. This transformation may introduce an important imine moiety for the analogues of trans-N-methyl-4-(1-naphthylvinyl)pyridine (NVP), a choline acetyltransferase (ChAT) inhibitor.
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http://dx.doi.org/10.1021/ct049890pDOI Listing
March 2005

Structural basis for androgen receptor interdomain and coactivator interactions suggests a transition in nuclear receptor activation function dominance.

Mol Cell 2004 Nov;16(3):425-38

Laboratories for Reproductive Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.

The androgen receptor (AR) is required for male sex development and contributes to prostate cancer cell survival. In contrast to other nuclear receptors that bind the LXXLL motifs of coactivators, the AR ligand binding domain is preferentially engaged in an interdomain interaction with the AR FXXLF motif. Reported here are crystal structures of the ligand-activated AR ligand binding domain with and without bound FXXLF and LXXLL peptides. Key residues that establish motif binding specificity are identified through comparative structure-function and mutagenesis studies. A mechanism in prostate cancer is suggested by a functional AR mutation at a specificity-determining residue that recovers coactivator LXXLL motif binding. An activation function transition hypothesis is proposed in which an evolutionary decline in LXXLL motif binding parallels expansion and functional dominance of the NH(2)-terminal transactivation domain in the steroid receptor subfamily.
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http://dx.doi.org/10.1016/j.molcel.2004.09.036DOI Listing
November 2004

Application of the PharmPrint methodology to two protein kinases.

J Chem Inf Comput Sci 2004 Sep-Oct;44(5):1803-9

Computational, Analytical, and Structural Sciences, GlaxoSmithKline, Five Moore Drive, Research Triangle Park, North Carolina 27709, USA.

The PharmPrint methodology developed by McGregor and Muskal1,2 was used to construct quantitative structure-activity relationship (QSAR) models for the prediction of cyclin-dependent kinase-2 (CDK2) and vascular endothelial growth factor receptor-2 (VEGFR2) inhibition. The QSAR models were constructed based on a binary description of biological activity--a value of zero for inactive and one for active compounds. Subsets of "active" kinase inhibitors (that is, inhibitors with pIC50 > or = 6.0) along with a subset of MDDR3 compounds serving as the recommended set of inactive compounds were used for model development. The predicted activities for the training set compounds were in excellent agreement with the assigned binary activities with greater than 92% of the compounds correctly classified. However, when the QSAR models were applied to the subsets of "inactive" kinase inhibitors (that is, inhibitors with pIC50 < 6.0), greater than 67% were incorrectly predicted to be active. Identical results were obtained with our CDK2 and VEGFR2 validation sets, where the majority of the inactive kinase inhibitors were predicted to be active. In efforts to improve the predictive performance of the QSAR models, simple, but important modifications were made to the PharmPrint methodology. On the basis of these modifications, a second set of QSAR models was constructed and applied to our validation sets to assess their predictive performance. Significant improvements were seen with the modified version of PharmPrint over the original. The results from both versions of PharmPrint are compared and discussed.
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http://dx.doi.org/10.1021/ci0498968DOI Listing
November 2005

Selection, application, and validation of a set of molecular descriptors for nuclear receptor ligands.

Comb Chem High Throughput Screen 2004 Aug;7(5):407-12

Computational, Analytical, and Structural Sciences, GlaxoSmithKline, Five Moore Drive, Research Triangle Park, NC 27709, USA.

A methodology for the selection and validation of nuclear receptor ligand chemical descriptors is described. After descriptors for a targeted chemical space were selected, a virtual screening methodology utilizing this space was formulated for the identification of potential NR ligands from our corporate collection. Using simple descriptors and our virtual screening method, we are able to quickly identify potential NR ligands from a large collection of compounds. As validation of the virtual screening procedure, an 8, 000-membered NR targeted set and a 24, 000-membered diverse control set of compounds were selected from our in-house general screening collection and screened in parallel across a number of orphan NR FRET assays. For the two assays that provided at least one hit per set by the established minimum pEC(50) for activity, the results showed a 2-fold increase in the hit-rate of the targeted compound set over the diverse set.
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http://dx.doi.org/10.2174/1386207043328535DOI Listing
August 2004

Structure and function of the glucocorticoid receptor ligand binding domain.

Vitam Horm 2004 ;68:49-91

Department of Gene Expression and Protein Biochemistry, Discovery Research, GlaxoSmithKline, Research Triangle Park, North Carolina 27709, USA.

After binding to an activating ligand, such as corticosteroid, the glucocorticoid receptor (GR) performs an impressive array of functions ranging from nuclear translocation, oligomerization, cofactor/kinase/transcription factor association, and DNA binding. One of the central functions of the receptor is to regulate gene expression, an activity triggered by ligand binding. In this role, GR acts as an adapter molecule by encoding the ligand's message within the structural flexibility of the ligand binding domain (LBD). The purpose of this review is to discuss the many structural and functional features of the GR LBD in light of recent successful biochemical and crystallographic studies. Progress in this area of research promises to reveal new strategies and insights allowing for the design of novel drugs to treat inflammatory diseases, diabetic conditions, steroid resistance, and cancers.
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http://dx.doi.org/10.1016/S0083-6729(04)68002-2DOI Listing
August 2004

Progesterone receptor ligand binding pocket flexibility: crystal structures of the norethindrone and mometasone furoate complexes.

J Med Chem 2004 Jun;47(13):3381-7

GlaxoSmithKline Inc., 5 Moore Drive, Research Triangle Park, North Carolina 27709, USA.

Although progesterone, the natural ligand of the progesterone receptor (PR), has a hydrogen atom at the 17alpha position, other potent steroid agonists such as norethindrone and mometasone furoate have larger substituents at this position that are accommodated by the PR ligand binding pocket. Crystallographic analysis of PR ligand binding domain complexes clearly demonstrated that these moieties were accommodated by local shifts of the protein main chain and by adoption of alternative side chain rotamer conformations of ligand-proximal amino acids. These conformational changes imparted a ligand-specific volume to the binding pocket, from 490 A3 in the metribolone complex to 520 A3 in the norethindrone complex, 565 A3 in the progesterone complex, and 730 A3 in the mometasone furoate complex. Despite these marked alterations in binding pocket volume, critical interactions essential for establishment of an active AF2 conformation were maintained.
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http://dx.doi.org/10.1021/jm030640nDOI Listing
June 2004

A new series of estrogen receptor modulators that display selectivity for estrogen receptor beta.

J Med Chem 2002 Dec;45(25):5492-505

GlaxoSmithKline Research and Development, Research Triangle Park, North Carolina 27709, USA.

A series of 1,3,5-triazine-based estrogen receptor (ER) modulators that are modestly selective for the ERbeta subtype are reported. Compound 1, which displayed modest potency and selectivity for ERbeta vs ERalpha, was identified via high-throughput screening utilizing an ERbeta SPA-based binding assay. Subsequent analogue preparation resulted in the identification of compounds such as 21 and 43 that display 25- to 30-fold selectivity for ERbeta with potencies in the 10-30 nM range. These compounds profile as full antagonists at ERbeta and weak partial agonists at ERalpha in a cell-based reporter gene assay. In addition, the X-ray crystal structure of compound 15 complexed with the ligand binding domain of ERbeta has been solved and was utilized in the design of more conformationally restrained analogues such as 31 in an attempt to increase selectivity for the ERbeta subtype.
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http://dx.doi.org/10.1021/jm020291hDOI Listing
December 2002

Crystal structure of the glucocorticoid receptor ligand binding domain reveals a novel mode of receptor dimerization and coactivator recognition.

Cell 2002 Jul;110(1):93-105

Gene Expression and Protein Biochemistry, Research Triangle Park, NC 27709, USA.

Transcriptional regulation by the glucocorticoid receptor (GR) is mediated by hormone binding, receptor dimerization, and coactivator recruitment. Here, we report the crystal structure of the human GR ligand binding domain (LBD) bound to dexamethasone and a coactivator motif derived from the transcriptional intermediary factor 2. Despite structural similarity to other steroid receptors, the GR LBD adopts a surprising dimer configuration involving formation of an intermolecular beta sheet. Functional studies demonstrate that the novel dimer interface is important for GR-mediated activation. The structure also reveals an additional charge clamp that determines the binding selectivity of a coactivator and a distinct ligand binding pocket that explains its selectivity for endogenous steroid hormones. These results establish a framework for understanding the roles of protein-hormone and protein-protein interactions in GR signaling pathways.
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http://dx.doi.org/10.1016/s0092-8674(02)00817-6DOI Listing
July 2002