Microcanonical insights into the physicochemical stability of the coformulation of insulin with amylin analogues.

Injections of insulin are the main treatment for diabetes, but in the long run this therapy can induce serious drawbacks. This has inspired new drugs able to decrease insulin requirements. For instance, human amylin (hIAPP) is a small hormone cosecreted by pancreatic β-cells with insulin to which is a synergistic partner. However, the high amyloidogenicity of hIAPP precluded it as a therapeutics and led to the design of pramlintide (sIAPP), a chimeric analogue with substitutions (A25P, S28P, and S29P) inherited from the aggregation-resistant rat isoform (rIAPP). Despite sIAPP advantages, it still shares with hIAPP a poorly soluble profile at physiological pH that hampers its mixture with insulin. Recent improvements, as charge-enhanced mutants, have been proposed. For instance, sIAPP+ was screened in silico by purely microcanonical thermostatistical methods and adds to sIAPP an S20R mutation to uplift its solubility. This suggests that such physically inspired computational approach may also be auspicious on devising effective coformulations of insulin with amylin analogues. In this seminal attempt, we make comparative multicanonical simulations of regular acting human insulin coformulated with hIAPP, sIAPP, or sIAPP+. To assess the respective physicochemical stabilities against aggregation, we characterize the structural-phase transitions through the microcanonical thermodynamic formalism and evaluate their time lags using the classical nucleation theory. These results are then correlated with estimates of solvation free energies, modeled by the Poisson-Boltzmann equation, and structural propensities. Experimental essays are compared to our simulations and support our methodology.