Simvastatin sodium salt and fluvastatin interact with the N-terminus of human gap junction gamma-3 protein Acknowledgements We thank EPSRC,Tangent Reprofiling Ltd (SME CASE Award) and the University of Warwick for funding and UHCW NHS Trust for access to facilities and patient tissue. References [1] S. J. Dilly, M. J. Bell, A. J. Clark, A. Marsh, R. M. Napier, M. J. Sergeant, A. J. Thompson, P. C. Taylor, Chem. Commun. 2007, 2808-2810; [2] A. Marsh, K. Casey-Green, S. J. Dilly S. James, W. Dimitri, S. R. Ladwa, P. C. Taylor, D. R. J. Singer PLOS ONE 2016 DOI: 10.1371/journal.pone.0148266; [3] S. Maeda, S. Nakagawa, M. Suga ,E. Yamashita, A. Oshima, Y. Fujiyoshi et al. Nature 458, 2009, 597-602; [4] W.-G. Liang, C.-C. Su, J.-H. Nian, A.-S. Chiang, S.-Y. Li, J.-J. Yang Cell Biochem Biophys 2011, 61, 189-197; [5] M. Billaud A. W. Lohman, S. R. Johnstone, L. A. Biwer S. Mutchler B. E. Isakson Pharmacol. Rev., 2014, 66, 513-569. Andrew Marsh,a* Katherine Casey-Green,a Fay Probert,a David Withall,a Daniel A. Mitchell,b Suzanne J. Dilly,d Sean James,e Wade Dimitri,e Sweta R. Ladwa,d Paul C. Taylor,a Donald R. J. Singerb,c,e** a Department of Chemistry, University of Warwick, Coventry, CV4 7AL b Clinical Sciences Research Institute, Warwick Medical School, Coventry, CV2 2DX c Fellowship of Postgraduate Medicine, 11 Chandos Street, London, W1G 9EB d Tangent Reprofiling Limited c/o SEEK, Central Point, 45 Beech St, London, EC2Y 8AD e University Hospital Coventry and Warwickshire NHS Trust, Coventry, CV2 2DX *@marshgroup a.marsh@warwick.ac.uk **@HealthMed As part of a programme to discover new targets of clinical therapeutics we have applied a chemical genomics approach[1] to the widely prescribed lipid-lowering pro-drug simvastatin. We prepared a genomic T7 phage library from mRNA isolated from sections of internal mammary artery obtained during cardiac bypass graft surgery (NHS National Research Ethics Service, Coventry Research Ethics Committee, Coventry UK: ref - 07/H1210/126).[2] Using immunohistochemistry we found GJC3 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 hydroxyacid. This interaction was also seen for fluvastatin. • Collected from patients in cardiac surgery at UHCW, Coventry • Transported in RNAse inhibitor solution and stored at –80°C • Extracted using Ambion MirVana miRNA isolation kit • 0.023% wt/wt from frozen tissue samples • Extracted using Novagen Straight A's mRNA extraction kit • 1.4% wt/wt from total RNA samples • cDNA prepared using Novagen OrientExpress cDNA kit • Engineered with digestion sites for phage vector ligation • cDNA ligated into T7Select® 10-3 phage vector • Phage amplified in E. coli BLT5615 • Initial library assayed to estimate diversity • Library amplified and a small number of clones sequenced Tissue sample Total RNA mRNA cDNA Phage vector Validation Storage • Library stored at 4°C initially • Long term storage of glycerol solutions stored at –80°C We studied interactions of clones of the bacteriophage, each expressing a different cardiovascular polypeptide, with surface-bound simvastatin that had been immobilised on 96-well plates using a validated photo- immobilisation method.[2] 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 (GJC3), also known as connexin 30.2/31.3 (mouse connexin Cx29).[2] Further analysis indicated the binding site to be the N-terminal domain (see homology model, orange highlight) putatively ‘regulating’ connexin hemichannel and gap junction pores. −30 −20 −10 0 10 0 25 50 75 100 125 150 175 200 225 250 275 residue number Hydropathy /kcalmol−1 protein GJC3 GJB2 pred