title: Engineering of insulin receptor isoform-selective insulin analogues.
abstract: BACKGROUND
  The insulin receptor (IR) exists in two isoforms, A and B, and the isoform expression pattern is tissue-specific. The C-terminus of the insulin B chain is important for receptor binding and has been shown to contact the IR just adjacent to the region where the A and B isoforms differ. The aim of this study was to investigate the importance of the C-terminus of the B chain in IR isoform binding in order to explore the possibility of engineering tissue-specific/liver-specific insulin analogues.
  
  
  RESULTS
  Insulin analogue libraries were constructed by total amino acid scanning mutagenesis. The relative binding affinities for the A and B isoform of the IR were determined by competition assays using scintillation proximity assay technology. Structural information was obtained by X-ray crystallography. Introduction of B25A or B25N mutations resulted in analogues with a 2-fold preference for the B compared to the A isoform, whereas the opposite was observed with a B25Y substitution. An acidic amino acid residue at position B27 caused an additional 2-fold selective increase in affinity for the receptor B isoform for analogues bearing a B25N mutation. Furthermore, the combination of B25H with either B27D or B27E also resulted in B isoform-preferential analogues (2-fold preference) even though the corresponding single mutation analogues displayed no differences in relative isoform binding affinity.
  
  
  CONCLUSIONS
  We have discovered a new class of IR isoform-selective insulin analogues with 2-4-fold differences in relative binding affinities for either the A or the B isoform of the IR compared to human insulin. Our results demonstrate that a mutation at position B25 alone or in combination with a mutation at position B27 in the insulin molecule confers IR isoform selectivity. Isoform-preferential analogues may provide new opportunities for developing insulin analogues with improved clinical benefits.
authors.affiliation: Diabetes Research Unit, Novo Nordisk A/S, Maaloev, Denmark. tgle@novonordisk.com
authors.forename: Tine
authors.initials: T
authors.lastname: Glendorf
authors.forename: Carsten E
authors.initials: CE
authors.lastname: Stidsen
authors.forename: Mathias
authors.initials: M
authors.lastname: Norrman
authors.forename: Erica
authors.initials: E
authors.lastname: Nishimura
authors.forename: Anders R
authors.initials: AR
authors.lastname: Sørensen
authors.forename: Thomas
authors.initials: T
authors.lastname: Kjeldsen
mesh_terms: D018360:Crystallography, X-Ray; D006801:Humans; D007328:Insulin / Q000031:analogs & derivatives / Q000378:metabolism*; D016296:Mutagenesis; D011487:Protein Conformation; D015202:Protein Engineering; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism*; D011972:Receptor, Insulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D007328:Insulin; D020033:Protein Isoforms; D011972:Receptor, Insulin