Marsh, Andrew, Casey-Green, Katherine, Probert, Fay, Withall, David M., Mitchell, Daniel A., Dilly , Suzanne J., James, Sean G., Dimitri, Wade, Ladwa, Sweta R. L., Taylor, Paul C. and Singer, Donald R. J. (2015) Data for Simvastatin sodium salt and fluvastatin interact with human gap junction gamma-3 protein. [Dataset]

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Abstract

Finding pleiomorphic targets for drugs allows new indications or warnings for treatment to be identified. As test of concept, we applied a new chemical genomics approach to uncover additional targets for the widely prescribed lipid-lowering pro-drug simvastatin. We used mRNA extracted from internal mammary artery from patients undergoing coronary artery surgery to prepare a viral cardiovascular protein library, using T7 bacteriophage. We then studied interactions of clones of the bacteriophage, each expressing a different cardiovascular polypeptide, with surface-bound simvastatin in 96-well plates. To maximise likelihood of identifying meaningful interactions between simvastatin and vascular peptides, we used a validated photo-immobilisation method to apply a series of different chemical linkers to bind simvastatin so as to present multiple orientations of its constituent components to potential targets. Three rounds of biopanning identified consistent interaction with the clone expressing part of the gene GJC3, which maps to Homo sapiens chromosome 7, and codes for gap junction gamma-3 protein, also known as connexin 30.2/31.3 (mouse connexin Cx29). Further analysis indicated the binding site to be for the N-terminal domain putatively ‘regulating’ connexin hemichannel and gap junction pores. Using immunohistochemistry we found connexin 30.2/ 31.3 to be present in samples of artery similar to those used to prepare the bacteriophage library. Surface plasmon resonance revealed that a 25 amino acid synthetic peptide representing the discovered N-terminus did not interact with simvastatin lactone, but did bind to the hydrolysed HMG CoA inhibitor, simvastatin acid. This interaction was also seen for fluvastatin. The gap junction blockers carbenoxolone and flufenamic acid also interacted with the same peptide providing insight into potential site of binding. These findings raise key questions about the functional significance of GJC3 transcripts in the vasculature and other tissues, and this connexin’s role in therapeutic and adverse effects of statins in a range of disease states.

Item Type:	Dataset
Subjects:	Q Science > QD Chemistry
Divisions:	Faculty of Science > Chemistry Faculty of Medicine > Warwick Medical School
Type of Data:	dideoxy sequencing; sequencing of phage; T-Coffee sequence alignment; SPR graphs; cell expression; Human Protein Database; Gap junction inhibitors; missense single nucleotide polymorphisms; insertion/deletion polymorphisms
Library of Congress Subject Headings (LCSH):	Cardiovascular system -- Disease -- Treatment, Statins (Cardiovascular agents), Connexins, Arteries
Publisher:	Department of Chemistry, University of Warwick
ISSN:	1932-6203
Official Date:	December 2015
Dates:	Date Event December 2015 Submitted
Status:	Not Peer Reviewed
Publication Status:	Published
Media of Output:	xlsx; docx; pdf; afasta; tif; rtf; txt
Access rights to Published version:	Open Access
Description:	S2 Dataset. The complete Sanger dideoxy sequencing data for a biopanning screen of simvastatin, immobilised from solution to a set 5 photochemistries on a 96-well polystyrene plate, versus a genomic T7 phage display created from samples of human vascular tissue total mRNA. S6 Dataset. Negative controls for sequencing of phage which bind to the same 5 photochemistries on a 96-well plate in the absence of simvastatin ligand. S9 Figure. T-Coffee (www.ebi.ac.uk) sequence alignment for GJC3 NP_853516.1 and GJB2 NP_003995.2. Graphical output in Kyte-Doolittle hydrophobicity colour scheme. S10 Dataset. T-Coffee aligned sequences in .afasta format for GJC3 NP_853516.1 and GJB2_NP003995.2. S16 Dataset. Complete set of SPR graphs as .xlsx spreadsheets. S18 Dataset. Normal tissue cell expression of gap junction protein, gamma 3, 30.2kDa (GJC3) analyzed using polyclonal antibody HPA015024. Accessed from Human Protein Atlas using keyword GJC3, July 2015 and saved as .xlsx spreadsheet S19 Dataset. The Human Protein Database annotates 19 gap junction proteins as connexins. http://www.proteinatlas.org/search/connexin with CNST, CAV3, CXADR data removed; exported as TAB data. Saved as .xslx file at http://figshare.com/articles/connexin_annotated_HTA_2015_xlsx/1422052 S20 Dataset. Gap junction inhibitors and known targets from ChEMBL_20 database. (Accessed 30 July 2015). S21 Dataset. GJC3 multiple sequence alignment for human, mouse and rat proteins showing how an alternative site of translation initiation results in extended N-terminus for mCx29. S23 Dataset. Human GJC3 missense single nucleotide polymorphisms from NCBI present in 1000 Genomes Project data (19 Dec 2015). S24 Dataset. Human GJC3 insertion/deletion polymorphisms from NCBI (19 Dec 2015).
RIOXX Funder/Project Grant:	Project/Grant ID RIOXX Funder Name Funder ID UNSPECIFIED [EPSRC] Engineering and Physical Sciences Research Council http://dx.doi.org/10.13039/501100000266 UNSPECIFIED University of Warwick http://dx.doi.org/10.13039/501100000741 UNSPECIFIED European Regional Development Fund http://dx.doi.org/10.13039/501100008530 UNSPECIFIED Birmingham Science City UNSPECIFIED UNSPECIFIED Advantage West Midlands (AWM) UNSPECIFIED UNSPECIFIED Tangent Reprofiling Ltd UNSPECIFIED
Related URLs:	Organisation Other Repository Related item in WRAP
Open Access Version:	Publisher

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