Novel GLP-1 receptor agonists offer hope for long-lasting diabetes treatment

By Dr. Sushama R. Chaphalkar, PhD.Reviewed by Benedette Cuffari, M.Sc.Oct 22 2023

In a study published in Scientific Reports, researchers engineer and computationally analyze three chimeric agonists of the glucagon-like peptide-1 (GLP-1) receptor by fusing native and mutant GLP-1 with designed ankyrin repeat protein (DARPin). This molecule binds human serum albumin (HSA). The fusion proteins were found to be stable, functional, and long-lasting, with high affinity for the GLP-1 receptor and human serum albumin, thus making them potential candidates for the treatment of type 2 diabetes mellitus (T2DM).

Study: Designing and computational analyzing of chimeric long-lasting GLP-1 receptor agonists for type 2 diabetes. Image Credit: myskin / Shutterstock.com

Background

Although various treatment modalities, including diet and lifestyle modification, antidiabetic medications, and insulin, are available to treat T2DM, glycemic control in individuals with diabetes remains poor, thus emphasizing the need for new treatment options.

GLP-1 and its analogs have shown promising results intreatingf T2DM in recent years; however, their therapeutic applicability is limited due to their short physiological half-lifeof lesss than two minutes. Therefore, there is a need to develop long-acting GLP-1 receptor agonists that resist proteolytic degradation and renal clearance.

Researchers in the present study aimed to address this need by fusing native GLP-1 and its protease-resistant mutants with DARPin, an HSA-binding protein with a longer half-life. The fusion proteins were studied in silico to assess their stability, function, and affinity towards specific targets.

About the study

To prevent recognition and degradation of GLP-1 by trypsin or dipeptidyl peptidase IV (DPP-IV), two protease-resistant GLP-1 mutants (mGLP-1) were developed by substituting specific amino acids in the GLP-1 sequence.

The native GLP-1 (nGLP-1) and mGLP-1 molecules were each genetically fused to the N-terminus of DARPin using a rigid, helical linker to create three fusion proteins, including nGLP-1-DARPin, mGLP-1-DARPin-1, and mGLP-1-DARPin-2. The rigid linker helped maintain the distance between the domains of the protein, thereby enabling the preservation of their individual biological functions. The fusion proteins were made of 168 amino acids encoded by 504 nucleotides.

Physical and chemical properties of the fusion proteins, including the molecular formula, molecular weight, number of charged residues, grand average hydropathy, aliphatic index, and instability index, were determined using the Expasy ProtParam server. While the secondary structures of the fusion proteins were predicted using SOPMA and PORTER servers, their three-dimensional (3D) structure was modeled using the trRosetta server.

The predicted structures were validated using the Ramachandran plot, ERRAT, and ProSA web server. The solubility of the proteins was assessed using the Protein-Sol web server.

The dynamic behavior of the three fusion proteins was studied using molecular dynamics (MD) simulations for 500 nanoseconds (ns) using the GROMACS tool. To understand the affinity of proteins to HSA and GLP-1’s extracellular domain, protein-protein docking simulations were performed using the ClusPro 2.0 server. The HSA-estimated binding affinities of the proteins were used as indicators of their prolonged half-life and slower renal clearance.

Study findings

The molecular weight of the fusion proteins was estimated to be about 17.5 kDa. The molecules were enriched in negatively charged amino acids as indicated by their isoelectric point.

The instability index and aliphatic index of the proteins indicated that they were stable and thermostable. The solubility and hydropathy scores of the proteins suggest that they were soluble upon expression. None of the fusion proteins were found to have toxic potential, as demonstrated by the results from the ToxDL server.

The proteins were primarily composed of alpha helices and random coils, while beta-turns and extended strands were absent. Nuclear magnetic resonance (NMR)-based structural analysis showed that the rigid linker used for fusion did not alter the structure or biological function of the fusion proteins as predicted by the 3D modeling tool. The results from the Ramachandran plot, ERRAT server, and ProSA server suggest that the quality of the predicted 3D models was good and comparable to native proteins.

The stability of the nGLP-1-DARPin protein was relatively lower than that of the mutant proteins. While the GLP-1 moiety of the structures was the most flexible region, all three proteins remained stable and compact while also retaining their biological activity, throughout the MD simulations.

Binding affinity studies using molecular docking revealed that the fusion proteins retained the ability to bind to the GLP-1 receptor as well as HSA. However, they showed greater affinity for HSA than the GLP-1 receptor.

The study highlights the use of albumin-binding proteins instead of albumin as fusion partners to improve the cost-effectiveness and safety of a fusion protein while prolonging its physiological half-life. The current study also demonstrates the utility of computational approaches and readily available bioinformatics tools in reducing the cost and time of future experimental studies while improving their success rate.

Conclusions

The long-lasting and protease-resistant chimeric proteins engineered in this study could potentially be developed into future therapeutics for T2DM patients. Further experimental and clinical research is required to confirm these findings.