Publications by authors named "Robert T Gampe"

13 Publications

  • Page 1 of 1

BacMam production and crystal structure of nonglycosylated apo human furin at 1.89 Å resolution.

Acta Crystallogr F Struct Biol Commun 2019 Apr 13;75(Pt 4):239-245. Epub 2019 Mar 13.

Platform Technology and Science, Department of Protein Cellular and Structural Sciences, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, PA 19426, USA.

Furin, also called proprotein convertase subtilisin/kexin 3 (PCSK3), is a calcium-dependent serine endoprotease that processes a wide variety of proproteins involved in cell function and homeostasis. Dysregulation of furin has been implicated in numerous disease states, including cancer and fibrosis. Mammalian cell expression of the furin ectodomain typically produces a highly glycosylated, heterogeneous protein, which can make crystallographic studies difficult. Here, the expression and purification of nonglycosylated human furin using the BacMam technology and site-directed mutagenesis of the glycosylation sites is reported. Nonglycosylated furin produced using this system retains full proteolytic activity indistinguishable from that of the glycosylated protein. Importantly, the nonglycosylated furin protein reliably forms extremely durable apo crystals that diffract to high resolution. These crystals can be soaked with a wide variety of inhibitors to enable a structure-guided drug-discovery campaign.
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http://dx.doi.org/10.1107/S2053230X19001419DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6450522PMC
April 2019

Discovery of GSK2656157: An Optimized PERK Inhibitor Selected for Preclinical Development.

ACS Med Chem Lett 2013 Oct 12;4(10):964-8. Epub 2013 Aug 12.

Oncology Research, Protein Dynamics DPU, GlaxoSmithKline Research and Development , Collegeville, Pennsylvania 19426, United States.

We recently reported the discovery of GSK2606414 (1), a selective first in class inhibitor of protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), which inhibited PERK activation in cells and demonstrated tumor growth inhibition in a human tumor xenograft in mice. In continuation of our drug discovery program, we applied a strategy to decrease inhibitor lipophilicity as a means to improve physical properties and pharmacokinetics. This report describes our medicinal chemistry optimization culminating in the discovery of the PERK inhibitor GSK2656157 (6), which was selected for advancement to preclinical development.
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http://dx.doi.org/10.1021/ml400228eDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4027568PMC
October 2013

Discovery of 7-methyl-5-(1-{[3-(trifluoromethyl)phenyl]acetyl}-2,3-dihydro-1H-indol-5-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine (GSK2606414), a potent and selective first-in-class inhibitor of protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK).

J Med Chem 2012 Aug 8;55(16):7193-207. Epub 2012 Aug 8.

Oncology Research, Protein Dynamics DPU, GlaxoSmithKline Research and Development, Collegeville, Pennsylvania 19426, United States.

Protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) is activated in response to a variety of endoplasmic reticulum stresses implicated in numerous disease states. Evidence that PERK is implicated in tumorigenesis and cancer cell survival stimulated our search for small molecule inhibitors. Through screening and lead optimization using the human PERK crystal structure, we discovered compound 38 (GSK2606414), an orally available, potent, and selective PERK inhibitor. Compound 38 inhibits PERK activation in cells and inhibits the growth of a human tumor xenograft in mice.
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http://dx.doi.org/10.1021/jm300713sDOI Listing
August 2012

Structure of Rev-erbalpha bound to N-CoR reveals a unique mechanism of nuclear receptor-co-repressor interaction.

Nat Struct Mol Biol 2010 Jul 27;17(7):808-14. Epub 2010 Jun 27.

Division of Endocrinology, Diabetes and Metabolism, Departments of Medicine and Genetics, The Institute for Diabetes, Obesity and Metabolism, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA.

Repression of gene transcription by the nuclear receptor Rev-erbalpha plays an integral role in the core molecular circadian clock. We report the crystal structure of a nuclear receptor-co-repressor (N-CoR) interaction domain 1 (ID1) peptide bound to truncated human Rev-erbalpha ligand-binding domain (LBD). The ID1 peptide forms an unprecedented antiparallel beta-sheet with Rev-erbalpha, as well as an alpha-helix similar to that seen in nuclear receptor ID2 crystal structures but out of register by four residues. Comparison with the structure of Rev-erbbeta bound to heme indicates that ID1 peptide and heme induce substantially different conformational changes in the LBD. Although heme is involved in Rev-erb repression, the structure suggests that Rev-erbalpha could also mediate repression via ID1 binding in the absence of heme. The previously uncharacterized secondary structure induced by ID1 peptide binding advances our understanding of nuclear receptor-co-repressor interactions.
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http://dx.doi.org/10.1038/nsmb.1860DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3719173PMC
July 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

Synthesis and biological activities of novel indole derivatives as potent and selective PPARgamma modulators.

Bioorg Med Chem Lett 2010 Feb 4;20(4):1399-404. Epub 2010 Jan 4.

Department of Medicinal Chemistry, Laboratoire GlaxoSmithKline, Centre de Recherches, 25-27 Avenue du Québec, 91951 Les Ulis, France.

Starting from the structure of Telmisartan, a new series of potent and selective PPARgamma modulators was identified. The synthesis, in vitro and in vivo evaluation of the most potent compounds are reported and the X-ray structure of compound 7b bound to the PPARgamma ligand binding domain is described.
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http://dx.doi.org/10.1016/j.bmcl.2009.12.107DOI Listing
February 2010

Modulation of androgen receptor activation function 2 by testosterone and dihydrotestosterone.

J Biol Chem 2007 Aug 25;282(35):25801-16. Epub 2007 Jun 25.

Curriculum in Toxicology, Laboratories for Reproductive Biology, Lineberger Comprehensive Cancer Center, Department of Pediatrics, University of North Carolina, Chapel Hill 27599, USA.

The androgen receptor (AR) is transcriptionally activated by high affinity binding of testosterone (T) or its 5alpha-reduced metabolite, dihydrotestosterone (DHT), a more potent androgen required for male reproductive tract development. The molecular basis for the weaker activity of T was investigated by determining T-bound ligand binding domain crystal structures of wild-type AR and a prostate cancer somatic mutant complexed with the AR FXXLF or coactivator LXXLL peptide. Nearly identical interactions of T and DHT in the AR ligand binding pocket correlate with similar rates of dissociation from an AR fragment containing the ligand binding domain. However, T induces weaker AR FXXLF and coactivator LXXLL motif interactions at activation function 2 (AF2). Less effective FXXLF motif binding to AF2 accounts for faster T dissociation from full-length AR. T can nevertheless acquire DHT-like activity through an AR helix-10 H874Y prostate cancer mutation. The Tyr-874 mutant side chain mediates a new hydrogen bonding scheme from exterior helix-10 to backbone protein core helix-4 residue Tyr-739 to rescue T-induced AR activity by improving AF2 binding of FXXLF and LXXLL motifs. Greater AR AF2 activity by improved core helix interactions is supported by the effects of melanoma antigen gene protein-11, an AR coregulator that binds the AR FXXLF motif and targets AF2 for activation. We conclude that T is a weaker androgen than DHT because of less favorable T-dependent AR FXXLF and coactivator LXXLL motif interactions at AF2.
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http://dx.doi.org/10.1074/jbc.M703268200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4075031PMC
August 2007

Crystallization of protein-ligand complexes.

Acta Crystallogr D Biol Crystallogr 2007 Jan 13;63(Pt 1):72-9. Epub 2006 Dec 13.

Department of Computational, Analytical and Structural Sciences, Glaxo SmithKline, 5 Moore Drive, Research Triangle Park, NC 27709, USA.

Obtaining diffraction-quality crystals has long been a bottleneck in solving the three-dimensional structures of proteins. Often proteins may be stabilized when they are complexed with a substrate, nucleic acid, cofactor or small molecule. These ligands, on the other hand, have the potential to induce significant conformational changes to the protein and ab initio screening may be required to find a new crystal form. This paper presents an overview of strategies in the following areas for obtaining crystals of protein-ligand complexes: (i) co-expression of the protein with the ligands of interest, (ii) use of the ligands during protein purification, (iii) cocrystallization and (iv) soaks.
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http://dx.doi.org/10.1107/S0907444906047020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2483499PMC
January 2007

Probing the functional link between androgen receptor coactivator and ligand-binding sites in prostate cancer and androgen insensitivity.

J Biol Chem 2006 Mar 19;281(10):6648-63. Epub 2005 Dec 19.

Laboratory for Reproductive Biology, Lineberger Comprehensive Cancer Center, Department of Pediatrics, Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599, USA.

The androgen receptor (AR) is a ligand-activated transcription factor required for male sex development and virilization and contributes to prostate cancer initiation and progression. High affinity androgen binding triggers conformational changes required for AR transactivation. Here we characterized naturally occurring AR gene mutations in the region of activation function 2 (AF2) that decrease or increase AR transcriptional activity by altering the region bounded by AF2 and the ligand binding pocket without affecting equilibrium androgen binding affinity. In the androgen insensitivity syndrome, germ line AR mutations increase the androgen dissociation rate and reduce AR FXXLF motif binding and the recruitment of steroid receptor coactivator (SRC)/p160 coactivator LXXLL motifs. In prostate cancer, somatic AR mutations in AF2 or near the bound ligand slow androgen dissociation and increase AR stabilization and coactivator recruitment. Crystal structures of the AR ligand binding domain bound to R1881 and FXXLF or LXXLL motif peptide indicate the mutations are proximal to the AF2 bound peptide, adjacent to the ligand pocket, or in a putative ligand gateway. The results suggest a bidirectional structural relay between bound ligand and coactivator that establishes AR functional potency in vivo.
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http://dx.doi.org/10.1074/jbc.M511738200DOI Listing
March 2006

Semicarbazone-based inhibitors of cathepsin K, are they prodrugs for aldehyde inhibitors?

Bioorg Med Chem Lett 2006 Feb 15;16(4):978-83. Epub 2005 Nov 15.

Department of Research Bioanalysis and Drug Metabolism, GlaxoSmithKline, Research Triangle Park, NC 27709, USA.

Starting from potent aldehyde inhibitors with poor drug properties, derivatization to semicarbazones led to the identification of a series of semicarbazone-based cathepsin K inhibitors with greater solubility and better pharmacokinetic profiles than their parent aldehydes. Furthermore, a representative semicarbazone inhibitor attenuated bone resorption in an ex vivo rat calvarial bone resorption model. However, based on enzyme inhibition comparisons at neutral pH, semicarbazone hydrolysis rates, and 13C NMR experiments, these semicarbazones probably function as prodrugs of aldehydes.
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http://dx.doi.org/10.1016/j.bmcl.2005.10.108DOI Listing
February 2006

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

Structure-based design of potent retinoid X receptor alpha agonists.

J Med Chem 2004 Apr;47(8):2010-29

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

A series of tetrahydrobenzofuranyl and tetrahydrobenzothienyl propenoic acids that showed potent agonist activity against RXRalpha were synthesized via a structure-based design approach. Among the compounds studied, 46a,b showed not only very good potency against RXRalpha (K(i) = 6 nM) but was also found to be greater than 167-fold selective vs RARalpha (K(i) > 1000 nM). This compound profiled out as a full agonist in a cell-based transient transfection assay (EC(50) = 3 nM). The two antipodes were separated via chiral chromatography, and 46b was found to be 40-fold more potent than 46a. Interestingly, cocrystallization of 46a,b with the RXRalpha protein generated a liganded structure whereby the (S)-antipode was found in the binding pocket. Given orally in db/db mice or ZDF rats, 46a,b showed a significant glucose-lowering effect and an increase in liver mass. Triglycerides decreased significantly in db/db mice but increased in the ZDF rats. A dose-dependent decrease of nonesterified free fatty acids was seen in ZDF rats but not in db/db mice. These differences indicate a species specific effect of RXR agonists on lipid metabolism.
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http://dx.doi.org/10.1021/jm030565gDOI Listing
April 2004