Stereochemical inversion as a route to improved biophysical properties of therapeutic peptides exemplified by glucagon

Abstract

Peptides and small proteins are attractive therapeutic candidates due to their inherent selectivity and limited off-target effects. Unfortunately, their potential is often hindered by unfavorable physicochemical properties. This is particularly true in the case of glucagon, a peptide indispensable in the treatment of life-threatening hypoglycemia. Glucagon displays extremely low solubility in physiological buffers and suffers chemical degradation when the pH is adjusted in either direction. Here we systematically examine site-specific stereochemical inversion as a means to enhance aqueous solubility and stability, yet not diminish bio-potency or pharmacodynamics. We report several analogs that maintain full biological activity with substantially increased aqueous solubility, and resistance to fibrillation. We conclude that D-amino acids offer an attractive option for biophysical optimization of therapeutic peptides.

Discovery of Hyperstable Noncanonical Plant-Derived Epidermal Growth Factor Receptor Agonist and Analogs

“…For our structure-relationship activity studies of bleogen pB1 loop 4, we used complementary positional Ala- and d -amino acid scans previously developed by our laboratory. ,,, Positional scans have found success in the structure–activity relationship studies of the EGF-like myxoma virus growth factor, TGF-α, and endothelin. ,,, The Ala-scan provides information on the relationship between biological activities and side-chain compositions. , In contrast, the d -amino acid scan has the advantage of retaining hydrodynamic properties of the analogs. More importantly, the different stereochemistries of the d - and l -configuration could reveal the importance of side-chain orientation as a receptor-contact amino acid. ,− …”

Synthesis, Optimization, and Biological Evaluation of Corrinated Conjugates of the GLP-1R Agonist Exendin-4

“…An additional highlight of Cbi is its water solubility (400 mg/mL), akin to glucose (450 mg/mL). Use of Cbi conjugation with drugs with physical properties not suited to drug development, such as glucagon − for rescue of hypoglycemia, offers a great scope for exploration.…”

Structural Refinement of Glucagon for Therapeutic Use

“…Bioactivity varied with the site selection and interestingly most d -analogs demonstrated substantially improved selectivity as assessed for activity at the homologous GLP-1 receptor . Select analogs with near-native in vitro bioactivity, and much enhanced solubility and stability achieved through single site d -substitution demonstrated comparable speed and ability to raise blood glucose . The structural mechanism by which substitution with d -amino acids can boost aqueous solubility remains undefined and of continued interest.…”

“…A deeper inspection of the centrally positioned Ser16 revealed that many substitutions were biologically well tolerated at this single site. 104 Of greater intrigue was the apparent increase in aqueous physiological solubility of the analogs with single site D-amino acid substitution at other sequence locations. D-Amino acids are known to destabilize helices with a magnitude strongly dependent on the bulkiness of the side chain.…”