Design of An Ultra-Fast Acting Insulin Analog using Unnatural Amino Acids - Replacement of PheB24 with Cyclohexanylalanine

Abstract

Insulin replacement therapy is central to the treatment of Type 1 diabetes mellitus (T1DM). Clinical trials have shown that tight glycemic control in T1DM leads to reduction in long-term diabetic complications. In an effort to better mimic the normal post-prandial pattern of pancreatic insulin release, meal-time insulin analogs were developed in the 1990s using standard mutagenesis. These analogs exhibit more rapid absorption from the subcutaneous depot due to destabilization of the zinc insulin hexamer. Unfortunately, such conventional analogs do not achieve the rapid “on-off” pharmacodynamic profiles sought in an ideal rapid-acting insulin formulation. To circumvent this limitation, we have investigated the utility of nonstandard protein design based on unnatural mutagenesis. Our studies have focused on residue PheB24 at the dimer interface of the insulin hexamer. Investigation of the role of this aromatic side chain by hybrid quantum-mechanical/classical molecular mechanics (QM/cMM) suggested that substitution of the B24 aromatic ring of its saturated near-isostere (cyclohexanylalanine; Cha) would introduce a subtle perturbation but still allow hexamer formation. We therefore undertook the characterization of a ChaB24-containing derivative of insulin lispro, the active component of Humalog. Native-like assembly of phenol-stabilized insulin analog hexamers was demonstrated in cobalt complexes by atomic absorption spectroscopy and explicitly visualized in zinc complexes by X-ray crystallography. ChaB24-lispro-insulin exhibits native hypoglycemic potency as measured in Lewis rats rendered diabetic by steptozotocin. Preliminary studies of pharmacodynamics in anesthetized Yorkshire pigs suggest that treatment of patients with ChaB24-lispro-insulin may benefit from curtailment of the late post-prandial “tail” of unwanted insulin action that presently confers risk of hypoglycemia. Together, these results exemplify the potential power of unnatural amino acids to optimize the therapeutic properties of a protein central to the treatment of a human disease.