A Light Activated Glucagon Trimer with Resistance to Fibrillation

Abstract

In this work, we have brought the release of glucagon under the control of light. The aim of this approach is to allow minimally invasive, two-hormone control of blood glucose. Glucagon has two major challenges associated with its therapeutic application: (1) the required amount and timing of glucagon release is highly variable, and (2) glucagon rapidly fibrillates in solution, forming aggregates that are inactive. We have developed a light activated glucagon trimer, in which we have joined three glucagon molecules via light cleaved linkers. We demonstrated that this material can be stimulated by light to release glucagon in a predictable manner. In addition, we demonstrated that in the absence of light, the trimer does not form fibrils and thus releases normal unfibrillated glucagon upon irradiation. These qualities make this material ideal for incorporation into a two hormone light-activated artificial pancreas system.

A Green Light-Activated Insulin Depot with Ultrafast In Vivo Efficiency in the Subcutaneous Space

“…Previous experience suggested that the three products correspond to the three amino groups present in insulin and that the major isomer-3 was due to the modification of the sole lysine present (previously shown to be the most reactive site). 17,21 To confirm the nature of isomer-3, we subjected it to proteolysis via Glu-C, which cleaves on the carboxyl side of the glutamic acid residues. In HPLC we observed four main products, labeled 1 through 4 (Figure 4).…”

Fuzzy Drug Targets: Disordered Proteins in the Drug-Discovery Realm

“…Interaction with disordered macromolecules will retain the polarization of the emitted light. Fluorescence polarization also allows HTS and therefore is a useful technique in IDP drug discovery. , Aggregation inhibitors may be screened using fluorescence-based assays for the determination of aggregation kinetics, which is an easy and inexpensive approach for HTS . While CD spectroscopy can help determine the drug-induced changes in IDP structure, its use is limited by its sensitivity.…”

“… 86 , 87 Aggregation inhibitors may be screened using fluorescence-based assays for the determination of aggregation kinetics, which is an easy and inexpensive approach for HTS. 88 While CD spectroscopy can help determine the drug-induced changes in IDP structure, its use is limited by its sensitivity. CD is also sensitive to the types of buffers and salts used, making its adoption difficult for HTS.…”

Strategies for Conditional Regulation of Proteins

“…The enzymatic active site or a binding interface of a POI are the target sites for installation of caging groups. Decaging occurs when these functional groups are exposed to specific physical or chemical stimuli including small molecules, light, metal ions, or enzymes. − Unnatural amino acids (UAAs) present bioorthogonal functionalities for easy and site-specific incorporation of caging groups. Alternatively, caged residues can be directly introduced at desired sites in the POI using genetic code expansion (GCE) and specific stimuli can be used to decage these residues on demand. − UAAs were also introduced in proteins to permit cleavage of the peptide backbone at specific sites with an appropriate stimulus .…”