Publications by authors named "John J Kerrigan"

8 Publications

  • Page 1 of 1

Development of a high-throughput screen to detect inhibitors of TRPS1 sumoylation.

Assay Drug Dev Technol 2013 Jun;11(5):308-25

Molecular Discovery Research, GlaxoSmithKline, Collegeville, PA 19426, USA.

Small ubiquitin-like modifier (SUMO) belongs to the family of ubiquitin-like proteins (Ubls) that can be reversibly conjugated to target-specific lysines on substrate proteins. Although covalently sumoylated products are readily detectible in gel-based assays, there has been little progress toward the development of robust quantitative sumoylation assay formats for the evaluation of large compound libraries. In an effort to identify inhibitors of ubiquitin carrier protein 9 (Ubc9)-dependent sumoylation, a high-throughput fluorescence polarization assay was developed, which allows detection of Lys-1201 sumoylation, corresponding to the major site of functional sumoylation within the transcriptional repressor trichorhino-phalangeal syndrome type I protein (TRPS1). A minimal hexapeptide substrate peptide, TMR-VVK₁₂₀₁TEK, was used in this assay format to afford high-throughput screening of the GlaxoSmithKline diversity compound collection. A total of 728 hits were confirmed but no specific noncovalent inhibitors of Ubc9 dependent trans-sumoylation were found. However, several diaminopyrimidine compounds were identified as inhibitors in the assay with IC₅₀ values of 12.5 μM. These were further characterized to be competent substrates which were subject to sumoylation by SUMO-Ubc9 and which were competitive with the sumoylation of the TRPS1 peptide substrates.
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http://dx.doi.org/10.1089/adt.2012.501DOI Listing
June 2013

Understanding the origins of time-dependent inhibition by polypeptide deformylase inhibitors.

Biochemistry 2011 Aug;50(31):6642-54

Department of Biological Reagents, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, Pennsylvania 19426, USA.

The continual bacterial adaptation to antibiotics creates an ongoing medical need for the development of novel therapeutics. Polypeptide deformylase (PDF) is a highly conserved bacterial enzyme, which is essential for viability. It has previously been shown that PDF inhibitors represent a promising new area for the development of antimicrobial agents, and that many of the best PDF inhibitors demonstrate slow, time-dependent binding. To improve our understanding of the mechanistic origin of this time-dependent inhibition, we examined in detail the kinetics of PDF catalysis and inhibition by several different PDF inhibitors. Varying pH and solvent isotope led to clear changes in time-dependent inhibition parameters, as did inclusion of NaCl, which binds to the active site metal of PDF. Quantitative analysis of these results demonstrated that the observed time dependence arises from slow binding of the inhibitors to the active site metal. However, we also found several metal binding inhibitors that exhibited rapid, non-time-dependent onset of inhibition. By a combination of structural and chemical modification studies, we show that metal binding is only slow when the rest of the inhibitor makes optimal hydrogen bonds within the subsites of PDF. Both of these interactions between the inhibitor and enzyme were found to be necessary to observe time-dependent inhibition, as elimination of either leads to its loss.
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http://dx.doi.org/10.1021/bi200655gDOI Listing
August 2011

Production of protein complexes via co-expression.

Protein Expr Purif 2011 Jan 6;75(1):1-14. Epub 2010 Aug 6.

Biological Reagents & Assay Development, Platform Technology & Science, GlaxoSmithKline R&D, 1250 South Collegeville Road, Collegeville, PA 19426, USA.

Multi-protein complexes are involved in essentially all cellular processes. A protein's function is defined by a combination of its own properties, its interacting partners, and the stoichiometry of each. Depending on binding partners, a transcription factor can function as an activator in one instance and a repressor in another. The study of protein function or malfunction is best performed in the relevant context. While many protein complexes can be reconstituted from individual component proteins after being produced individually, many others require co-expression of their native partners in the host cells for proper folding, stability, and activity. Protein co-expression has led to the production of a variety of biological active complexes in sufficient quantities for biochemical, biophysical, structural studies, and high throughput screens. This article summarizes examples of such cases and discusses critical considerations in selecting co-expression partners, and strategies to achieve successful production of protein complexes.
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http://dx.doi.org/10.1016/j.pep.2010.07.015DOI Listing
January 2011

Optimized procedures for producing biologically active chemokines.

Protein Expr Purif 2009 Jun;65(2):251-60

GlaxoSmithKline, Biological Reagents & Assay Development, Mail Code: UE0548, 709 Swedeland Road, King of Prussia, PA 19406, USA.

We describe here two strategies to produce biologically active chemokines with authentic N-terminal amino acid residues. The first involves producing the target chemokine with an N-terminal 6xHis-SUMO tag in Escherichia coli as inclusion bodies. The fusion protein is solubilized and purified with Ni-NTA-agarose in denaturing reagents. This is further followed by tag removal and refolding in a redox refolding buffer. The second approach involves expressing the target chemokine with an N-terminal 6xHis-Trx-SUMO tag in an engineered E. coli strain that facilitates formation of disulfide bonds in the cytoplasm. Following purification of the fusion protein via Ni-NTA and tag removal, the target chemokine is refolded without redox buffer and purified by reverse phase chromatography. Using the procedures, we have produced more than 15 biologically active chemokines, with a yield of up to 15 mg/L.
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http://dx.doi.org/10.1016/j.pep.2009.01.017DOI Listing
June 2009

Frameshift events associated with the lysyl-tRNA and the rare arginine codon, AGA, in Escherichia coli: a case study involving the human Relaxin 2 protein.

Protein Expr Purif 2008 Aug 5;60(2):110-6. Epub 2008 Mar 5.

Department of Biological Reagents and Assay Development, GlaxoSmithKline, 709 Swedeland Road, King of Prussia, PA 19406, USA.

Human Relaxin 2 is an insulin-related peptide hormone with a mass of 19,084 Da. The mRNA contains a number of arginine codons that are rarely used by Escherichia coli to produce highly expressed proteins. As a result, expressing this recombinant protein in E. coli is problematic. When human Relaxin 2 was expressed in E. coli BL21 (DE3), several forms of the protein were made. One species had the expected molecular weight (19,084 Da). A second species observed had a molecular weight of 21,244 Da. A third minor species had a molecular weight of 17,118 Da. These aberrant molecular weights can be explained as follows. First, a sequence CGA-AAA-AAG-AGA, containing the rare arginine codons CGA and AGA was the site of the +1 frameshift that generated the 21,244 Da species. Since there was a limited supply of this arginyl-tRNA, the peptidyl-tRNA moved +1 nucleotide to occupy the codon and resumed protein synthesis. Second, a -1 frameshift associated with 'slippery A' sequence XXA-AAA-AAG accounted for 10% of the product with a mass of 17,118 Da. Presumably, the shift to -1 also occurred because there was a paucity of the arginyl-tRNAArgucu. Introduction of a plasmid coding for the cognate tRNA for AGA and site directed mutagenesis prevented the formation of both frameshift species.
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http://dx.doi.org/10.1016/j.pep.2008.02.016DOI Listing
August 2008

Suppressing posttranslational gluconoylation of heterologous proteins by metabolic engineering of Escherichia coli.

Appl Environ Microbiol 2008 Feb 14;74(4):950-8. Epub 2007 Dec 14.

Microbial and Cell Culture Development, GlaxoSmithKline, 709 Swedeland Road, King of Prussia, PA 19406, USA.

Minimization of chemical modifications during the production of proteins for pharmaceutical and medical applications is of fundamental and practical importance. The gluconoylation of heterologously expressed protein which is observed in Escherichia coli BL21(DE3) constitutes one such undesired posttranslational modification. We postulated that formation of gluconoylated/phosphogluconoylated products of heterologous proteins is caused by the accumulation of 6-phosphogluconolactone due to the absence of phosphogluconolactonase (PGL) in the pentose phosphate pathway. The results obtained demonstrate that overexpression of a heterologous PGL in BL21(DE3) suppresses the formation of the gluconoylated adducts in the therapeutic proteins studied. When this E. coli strain was grown in high-cell-density fed-batch cultures with an extra copy of the pgl gene, we found that the biomass yield and specific productivity of a heterologous 18-kDa protein increased simultaneously by 50 and 60%, respectively. The higher level of PGL expression allowed E. coli strain BL21(DE3) to satisfy the extra demand for precursors, as well as the energy requirements, in order to replicate plasmid DNA and express heterologous genes, as metabolic flux analysis showed by the higher precursor and NADPH fluxes through the oxidative branch of the pentose phosphate shunt. This work shows that E. coli strain BL21(DE3) can be used as a host to produce three different proteins, a heterodimer of liver X receptors, elongin C, and an 18-kDa protein. This is the first report describing a novel and general strategy for suppressing this nonenzymatic modification by metabolic pathway engineering.
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http://dx.doi.org/10.1128/AEM.01790-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2258596PMC
February 2008

Phylogenomic and biochemical characterization of three Legionella pneumophila polypeptide deformylases.

J Bacteriol 2006 Jul;188(14):5249-57

Microbiology Department, GlaxoSmithKline, 1250 S. Collegeville Road, Collegeville, PA 19426, USA.

Legionella pneumophila is a gram-negative facultative intracellular human pathogen that can cause fatal Legionnaires' disease. Polypeptide deformylase (PDF) is a novel broad-spectrum antibacterial target, and reports of inhibitors of PDF with potent activities against L. pneumophila have been published previously. Here, we report the identification of not one but three putative pdf genes, pdfA, pdfB, and pdfC, in the complete genome sequences of three strains of L. pneumophila. Phylogenetic analysis showed that L. pneumophila PdfA is most closely related to the commonly known gamma-proteobacterial PDFs encoded by the gene def. PdfB and PdfC are more divergent and do not cluster with any specific bacterial or eukaryotic PDF. All three putative pdf genes from L. pneumophila strain Philadelphia 1 have been cloned, and their encoded products have been overexpressed in Escherichia coli and purified. Enzymatic characterization shows that the purified PDFs with Ni2+ substituted are catalytically active and able to remove the N-formyl group from several synthetic polypeptides, although they appear to have different substrate specificities. Surprisingly, while PdfA and PdfB with Zn2+ substituted are much less active than the Ni2+ forms of each enzyme, PdfC with Zn2+ substituted was as active as the Ni2+ form for the fMA substrate and exhibited substrate specificity different from that of Ni2+ PdfC. Furthermore, the catalytic activities of these enzymes are potently inhibited by a known small-molecule PDF inhibitor, BB-3497, which also inhibits the extracellular growth of L. pneumophila. These results indicate that even though L. pneumophila has three PDFs, they can be effectively inhibited by PDF inhibitors which can, therefore, have potent anti-L. pneumophila activity.
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http://dx.doi.org/10.1128/JB.00866-05DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1539947PMC
July 2006

Discovery of substituted maleimides as liver X receptor agonists and determination of a ligand-bound crystal structure.

J Med Chem 2005 Aug;48(17):5419-22

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

Substituted 3-(phenylamino)-1H-pyrrole-2,5-diones were identified from a high throughput screen as inducers of human ATP binding cassette transporter A1 expression. Mechanism of action studies led to the identification of GSK3987 as an LXR ligand. GSK3987 recruits the steroid receptor coactivator-1 to human LXRalpha and LXRbeta with EC(50)s of 40 nM, profiles as an LXR agonist in functional assays, and activates LXR though a mechanism that is similar to first generation LXR agonists.
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http://dx.doi.org/10.1021/jm050532wDOI Listing
August 2005