Synthesis and biological activity of seventeen analogues of human insulin

We synthesized seventeen analogues of human insulin, applying the principle of stepwise, selective formation of the disulphide bonds. Most of these analogues only differ from human insulin in the replacement of a single amino acid in positions 2, 5, 6, 7, 8 and 11 of the A chain and 5, 7, 13 and 16 of the B-chain. The influence of these modifications on the physicochemical properties of the analogues is discussed. Eight analogues could be crystallized. All the analogues produce the same biological effects as insulin, but differ markedly in their potency. In isolated fat cells in vitro, [HisA8]insulin showed a relative potency of 2.46 in stimulating glucose oxidation (human insulin = 1), whereas [D-CysA6,A11]insulin had a potency of only 0.00027. Very low potency was observed when IleA2 or the half-cystines A6, A7, A11 or B7 were modified. Replacement of the invariant GlnA5 by alanine only reduced potency slightly. All the analogues are full agonists. The effects of the analogues on glucose oxidation and lipolysis are correlated, supporting the view that they are mediated by a common receptor on the fat-cell membrane. Hypoglycaemic potencies in the rat were similar to potencies in vitro. As expected, no correlation was demonstrable between antiserum binding--measured in the radioimmunoassay--and biological activity. Several results of this investigation are difficult to reconcile with the current view regarding the structure-activity relationship of insulin which appears to require further refinement.