Physicochemical basis for the rapid time‐action of Lys^B28Pro^B29‐insulin: Dissociation of a protein‐ligand complex

Abstract

The rate-limiting step for the absorption of insulin solutions after subcutaneous injection is considered to be the dissociation of self-associated hexamers to monomers. To accelerate this absorption process, insulin analogues have been designed that possess full biological activity and yet have greatly diminished tendencies to self-associate. Sedimentation velocity and static light scattering results show that the presence of zinc and phenolic ligands (m-cresol and/or phenol) cause one such insulin analogue, L y~~~~P r o~~~-h u m a n insulin (LysPro), to associate into a hexameric complex. Most importantly, this ligand-bound hexamer retains its rapid-acting pharmacokinetics and pharmacodynamics. The dissociation of the stabilized hexameric analogue has been studied in vitro using static light scattering as well as in vivo using a female pig pharmacodynamic model. Retention of rapid time-action is hypothesized to be due to altered subunit packing within the hexamer. Evidence for modified monomer-monomer interactions has been observed in the X-ray crystal structure of a zinc LysPro hexamer (Ciszak E et al., 1995, Structure 3:615-622). The solution state behavior of LysPro, reported here, has been interpreted with respect to the crystal structure results. In addition, the phenolic ligand binding differences between LysPro and insulin have been compared using isothermal titrating calorimetry and visible absorption spectroscopy of cobalt-containing hexamers. These studies establish that rapid-acting insulin analogues of this type can be stabilized in solution via the formation of hexamer complexes with altered dissociation properties.

Biophysical Optimization of a Therapeutic Protein by Nonstandard Mutagenesis

“…The slight attenuation of this feature in the spectrum of the KP-insulin hexamer (thin black line in Fig. 6B; 12 Ϯ 1% lower at 574 nm in triplicate measurements) in relation to the wild-type hexamer (thick black line) has previously been described (9). The visible absorption spectrum of 3-I-Tyr B26 -KP-insulin as a metal-ionstabilized complex (open circles in Fig.…”

“…The molecular elegance of rapid-acting insulin analogs has come at a biophysical cost; such formulations are more susceptible to physical and chemical degradation (especially on dilution and above room temperature) 4 than is wild-type insulin (8,9). Such degradation reflects a trade-off intrinsic to the logic of their design as the same amino acid substitutions that enable accelerated hexamer disassembly in the subcutaneous depot also undermine the protective effects of these assemblies against degradation in the vial or delivery device.…”

“…1C) and a hexameric template wherein Tyr B26 packs at an aromatic-rich dimer interface (in the presence of Zn 2ϩ and phenol, metacresol, or cyclohexanol; Fig. 1, D and E) (9). The availability of these alternative templates facilitated comparative studies of biophysical properties pertinent to formulation and biological activity, including the TR allosteric transition 6 ( Fig.…”

“…These qualitative features do not exclude local reorganization of the B26-related substructure, determining the details of this structure will require more complete 1 H NMR analysis and molecular modeling. Insulin Self-assembly-Evidence that 3-I-Tyr B26 -KP-insulin and KP-insulin are each competent to form R 6 phenol-stabilized Co 2ϩ -insulin hexamers 9 was provided by observation of a characteristic d-d visible absorption band in each case. This blue band, which provides an optical signature of a tetrahedral binding site (Fig.…”

“…This blue band, which provides an optical signature of a tetrahedral binding site (Fig. 6A), is prominently observed in the spectrum 9 Insulin is capable of coordinating a variety of divalent metal ions to form hexamers in which the three His B10 side chains in each component trimer provide a coordination site (octahedral in T 3 sites and tetrahedral in R 3 or R f 3 sites) (1, 61). (Table 1) .…”