Physicochemical basis for the rapid time-action of LysB28ProB29-insulin: dissociation of a protein-ligand complex.
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, LysB28ProB29-human 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.
Bakaysa
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DL
Biopharmaceutical Development Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285, USA.
Radziuk
J
J
Havel
H A
HA
Brader
M L
ML
Li
S
S
Dodd
S W
SW
Beals
J M
JM
Pekar
A H
AH
Brems
D N
DN
D000494:Allosteric Regulation; D005260:Female; D006801:Humans; D007328:Insulin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D061268:Insulin Lispro; D008024:Ligands; D011485:Protein Binding; D011487:Protein Conformation
D007328:Insulin; D061268:Insulin Lispro; D008024:Ligands