Insulin analog with additional disulfide bond has increased stability and preserved activity

Abstract

Insulin is a key hormone controlling glucose homeostasis. All known vertebrate insulin analogs have a classical structure with three 100% conserved disulfide bonds that are essential for structural stability and thus the function of insulin. It might be hypothesized that an additional disulfide bond may enhance insulin structural stability which would be highly desirable in a pharmaceutical use. To address this hypothesis, we designed insulin with an additional interchain disulfide bond in positions A10/B4 based on Ca-Ca distances, solvent exposure, and side-chain orientation in human insulin (HI) structure. This insulin analog had increased affinity for the insulin receptor and apparently augmented glucodynamic potency in a normal rat model compared with HI. Addition of the disulfide bond also resulted in a 34.6°C increase in melting temperature and prevented insulin fibril formation under high physical stress even though the C-terminus of the Bchain thought to be directly involved in fibril formation was not modified. Importantly, this analog was capable of forming hexamer upon Zn addition as typical for wild-type insulin and its crystal structure showed only minor deviations from the classical insulin structure. Furthermore, the additional disulfide bond prevented this insulin analog from adopting the R-state conformation and thus showing that the R-state conformation is not a prerequisite for binding to insulin receptor as previously suggested. In summary, this is the first example of an insulin analog featuring a fourth disulfide bond with increased structural stability and retained function.

Development of glucose-responsive ‘smart’ insulin systems

“…Two classes of insulin analogs have been shown to be refractory to fibrillation: single-chain insulins (SCIs) containing a foreshortened C domain (Fig. 1A) (54) and two-chain insulin analogs containing an engineered non-canonical disulfide bridge between the A and B chains (55) (Fig. 1B).…”

“…In each case the altered topology (connectivity) of the polypeptide is thought to be incompatible with regular cross-β assembly, the general structural mechanism underlying formation of amyloid (56-58). Although such analogs have not been tested clinically, insertion of a non-canonical cystine is associated in rat studies with anomalously prolonged duration of activity on intravenous bolus injection (55); this would be an unfavorable feature of a pump insulin.…”

“…(PDB ID: 2LWZ) (B) 4SS-insulin is a two-chain insulin analog (A-chain, dark gray, B-chain, light gray) that contains an engineered fourth disulfide linkage (red asterisk) between the A- and B-chains in addition to its three native disulfide linkages (yellow). The analog was reported to be resistant to fibrillation and have increased temperature stability (55). (PDB ID: 4EFX)…”

Insulin‐like genes in ascidians: Findings in Ciona and hypotheses on the evolutionary origins of the pancreas

“…Among the main conserved residues are a few cysteines that establish intra‐ and inter‐chain disulfide bonds. Disulfide bonds are necessary for the stability and the biological activity of insulin and its related peptides (Vinther et al ., ), and enable them to control a wide range of cellular processes, including nutrient homeostasis and regulation of cell growth and survival in a variety of tissues, as well as aging (Edmondson et al ., ; Fulzele and Clemens, ; Kenyon, ; Werner and Leroith, ). In widely different organisms, cells producing insulin and related peptides have been described as specialized neurons in the nervous system, both central and peripheral, or as endocrine cells that can be either disseminated in various regions of the digestive tract or concentrated in the parenchyma of the pancreas or its evolutionary equivalents.…”

Conformational Dynamics Associated with Ligand Binding to Vertebrate Hexa-coordinate Hemoglobins

“…pointed out that disruption of disulfide bonds usually destabilize the native structure of proteins (Li et al, 2013;Vinther et al, 2013;Zhang et al, 2011b). The value of the m parameter, which is related to the exposure of the protein surface area to the solvent, was comparable for Cygb and Cygb red , whereas a slightly larger value was determined for CNCygb, suggesting an increase in the solvent-exposed surface area for the CN -bound form of Cygb.…”