Simvastatin Sodium Salt and Fluvastatin Interact with Human Gap Junction Gamma-3 Protein

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

Data for Simvastatin sodium salt and fluvastatin interact with human gap junction gamma-3 protein

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

Alignment of discovered contig29 versus GJC3 (residues 1–25) and the sequence for GJB2 (residues 1–25).

<p>Carried out using T-Coffee (<a href="http://www.ebi.ac.uk/" target="_blank">www.ebi.ac.uk</a>) with GJC3 NCBI sequence NP_853516.1 and the protein databank file corresponding to GJB2 code 2zw3.pdb. Consensus residues are shown in the upper row; figure prepared using UCSF <i>Chimera</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148266#pone.0148266.ref050" target="_blank">50</a>].</p

Preparation of a T7 phage display library from vascular tissue.

<p>Preparation of a T7 phage display library from vascular tissue.</p

Comparison of Kyte-Doolittle index hydropathy plot of GJC3 (orange) and GJB2 (blue) showing predicted transmembrane regions as positive values.

<p>Kyte-Doolittle analysis using ProtScale [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148266#pone.0148266.ref124" target="_blank">124</a>] <a href="http://web.expasy.org/protscale/" target="_blank">http://web.expasy.org/protscale/</a>. Parameters used were: window size = 9, relative weight 100%, linear model, not normalized. Data replotted using <i>ggplot2</i> in <i>R</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148266#pone.0148266.ref125" target="_blank">125</a>].</p

Wimley-White hydropathy plot of GJC3 (orange) and GJB2 (blue) showing predicted transmembrane regions as positive negative values.

<p>Wimley-White octanol-to-water scale smoothed values for GJC3 and GJB2 are shown, where positive hydropathy values indicate expected membrane associated residues. The predicted transmembrane (TM) segments indicated are at least 19 residues in length and were calculated using a validated algorithm [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148266#pone.0148266.ref126" target="_blank">126</a>]. Wimley-White smoothed hydropathy calculated using MPEx [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148266#pone.0148266.ref127" target="_blank">127</a>] <a href="http://blanco.biomol.uci.edu/mpex/" target="_blank">http://blanco.biomol.uci.edu/mpex/</a> and replotted using <i>ggplot2</i> in <i>R</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148266#pone.0148266.ref125" target="_blank">125</a>].</p

Alternative mRNA splicing and selected single nucleotide polymorphisms for <i>GJC3</i> products.

<p>CCDS entry CCDS34697.1 (accessed 24 May 2012) for transcribed mRNA from <i>GJC3</i> (NM_181538.2 and NP_853516.1) and its translated 279 amino acid polypeptide showing the alternate exon region, designated with a blue rectangle. Residues highlighted in purple (M1V, R4V, P19H, R22H, E268D) indicate single nucleotide polymorphisms (SNPs: see main text) leading to variation in coded amino acids within predicted geometrically neighbouring and functionally significant N- and C-terminal regions. V261 highlighted in orange indicates the C-terminal splice variant start point. Discovered residues correspond to amino acids 1–25. Underlined residues S227S, T234N highlighted in green indicate phosphorylation sites identified from PhosphositePlus and genomic SNPs.</p

Ethidium bromide stained agarose gel showing a sample of clones from the phage display library after PCR to amplify the insert DNA.

<p>Hyperladder in lanes 1 and 25 allows estimation of insert length. PCR controls in lanes 22, 23 and 24 (positive, positive and negative).</p

Immunohistochemistry for human artery with antibody against GJC3, hCx30.2/31.3.

<p>(A) H-86—sc-68376, Santa Cruz Biotechnology, Inc., vs. (B) control section, both micrographs at 20 x magnification. Brown staining in (A) indicates selective localisation of GJC3 in media and endothelium of the artery.</p

Helical wheel projections of: (A) discovered N-terminal residues 1–25 and (B) C-terminal residues 226–227.

<p>Serine 227 and threonine 234 subject to post-translational phosphorylation circled in red.</p