title: Simvastatin Sodium Salt and Fluvastatin Interact with Human Gap Junction Gamma-3 Protein.
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.
authors: Department of Chemistry, University of Warwick, Coventry, CV4 7AL, United Kingdom.
authors: Andrew
authors: A
authors: Marsh
authors: Department of Chemistry, University of Warwick, Coventry, CV4 7AL, United Kingdom.
authors: Katherine
authors: K
authors: Casey-Green
authors: Department of Chemistry, University of Warwick, Coventry, CV4 7AL, United Kingdom.
authors: Fay
authors: F
authors: Probert
authors: Department of Chemistry, University of Warwick, Coventry, CV4 7AL, United Kingdom.
authors: David
authors: D
authors: Withall
authors: Division of Metabolic and Vascular Health, Clinical Sciences Research Laboratories, Warwick Medical School, University of Warwick, Coventry, CV2 2DX, United Kingdom.
authors: Daniel A
authors: DA
authors: Mitchell
authors: Tangent Reprofiling Ltd, c/o SEEK, Central Point, 45 Beech Street, London, EC2Y 8AD, United Kingdom.
authors: Suzanne J
authors: SJ
authors: Dilly
authors: University Hospital Coventry and Warwickshire, Clifford Bridge Road, Coventry CV2 2DX, United Kingdom.
authors: Sean
authors: S
authors: James
authors: University Hospital Coventry and Warwickshire, Clifford Bridge Road, Coventry CV2 2DX, United Kingdom.
authors: Wade
authors: W
authors: Dimitri
authors: Tangent Reprofiling Ltd, c/o SEEK, Central Point, 45 Beech Street, London, EC2Y 8AD, United Kingdom.
authors: Sweta R
authors: SR
authors: Ladwa
authors: Department of Chemistry, University of Warwick, Coventry, CV4 7AL, United Kingdom.
authors: Paul C
authors: PC
authors: Taylor
authors: University Hospital Coventry and Warwickshire, Clifford Bridge Road, Coventry CV2 2DX, United Kingdom.|Fellowship of Postgraduate Medicine, 11 Chandos St, London W1G 9EB, United Kingdom.|Division of Metabolic and Vascular Health, Clinical Sciences Research Laboratories, Warwick Medical School, University of Warwick, Coventry, CV2 2DX, United Kingdom.
authors: Donald R J
authors: DR
authors: Singer
mesh_terms: D000595:Amino Acid Sequence; D017123:Bacteriophage T7 / Q000235:genetics*; D001711:Biotransformation; D017630:Connexins / Q000737:chemistry* / Q000235:genetics; D003331:Coronary Vessels / Q000737:chemistry* / Q000601:surgery; D005229:Fatty Acids, Monounsaturated / Q000737:chemistry*; D000077340:Fluvastatin; D015870:Gene Expression; D006801:Humans; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors / Q000737:chemistry*; D007211:Indoles / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics; D019151:Peptide Library; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry; D010597:Pharmacogenetics; D055668:Photochemical Processes; D011355:Prodrugs / Q000737:chemistry; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D016415:Sequence Alignment; D019821:Simvastatin / Q000031:analogs & derivatives / Q000737:chemistry*
chemical_list: D017630:Connexins; D005229:Fatty Acids, Monounsaturated; C464674:GJC3 protein, human; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D007211:Indoles; D009419:Nerve Tissue Proteins; D019151:Peptide Library; D010455:Peptides; D011355:Prodrugs; D011994:Recombinant Proteins; D000077340:Fluvastatin; C063287:simvastatin acid; D019821:Simvastatin