Side-Chain Proline-Based Polymers as Effective Inhibitors for In Vitro Aggregation of Insulin

Abstract

Insulin fibril formation is considered as the hallmark of several debilitating pathological conditions. To develop effective therapeutics that are able to control the amyloidogenesis process and inhibit fibril formation, herein we have designed a side-chain proline (Pro)-based homopolymer and block copolymers through the reversible addition−fragmentation chain transfer (RAFT) polymerization technique and further explored their obligatory role in the in vitro insulin fibrillation process. Using a variety of biophysical tools, including turbidity measurements, thioflavin T (ThT) fluorescence kinetics, tyrosine (Tyr) fluorescence study, Nile red (NR) fluorescence assay, dynamic light scattering (DLS) study, circular dichroism (CD) measurements, and isothermal titration calorimetry (ITC) techniques, we demonstrated that Pro-based polymers can significantly inhibit the insulin fibrillation process. Among them, the Pro-based homopolymer acts as the most potent inhibitor of insulin fibrillation as confirmed by ThT assay, CD study, and transmission electron microscopic (TEM) analysis. Tyrosine fluorescence measurements and NR fluorescence assay revealed that hydrophobic interactions are the crucial factor that mainly controls the inhibition process. Apart from hydrophobic interactions, polar interactions may also be responsible for the inhibition process as evaluated by ITC study.

Prion-derived tetrapeptide stabilizes thermolabile insulin via conformational trapping

“…Toward development of insulin formulations with lower amyloidogenicity, newer excipients as well as chemically modified insulin moieties are being reported. Recent studies depicted generation of thermostable insulin by adding polysialic acid and proline-based homopolymer at specific amino acid residues (Ghosh et al, 2020;Kabotso et al, 2020). Aromatic small molecules such as resveratrol and rosmarinic acid were shown to enhance the biophysical stability of native insulin while preserving its physiological activity by stabilizing the hexameric form or preventing dimer dissociation, thereby restricting the thermal unfolding of monomers (Pathak et al, 2020;Zheng and Lazo, 2018).…”

Side‐chain amino‐acid‐based polymers: self‐assembly and bioapplications

“…Apart from the above widespread applications, self-assembled amino-acid-based polymeric nanostructures have also shown enormous potential in several other clinical applications such as biosensing, bioimaging, mimicking enzymatic activity, 100 inhibiting fibrillization of proteins meant for neurological and nonneuropathic disorders etc. 101 Recently, our group has explored the applications of side-chain proline-based polymers in inhibiting the formation of insulin fibrils, which is considered a hallmark of diabetes. 101 Considering the fascinating features of selfassembled amino-acid-based polymeric nanostructures, numerous research groups have focused on applying these materials in the protein aggregation field.…”

“…101 Recently, our group has explored the applications of side-chain proline-based polymers in inhibiting the formation of insulin fibrils, which is considered a hallmark of diabetes. 101 Considering the fascinating features of selfassembled amino-acid-based polymeric nanostructures, numerous research groups have focused on applying these materials in the protein aggregation field. 102,103 In this regard, Debnath et al have fabricated biopolymeric micelles that contained a polyaspartic acid backbone along with oleyl amine, ethylenediamine and arginine functional groups; the synthesized biopolymeric micelles clear the amyloid aggregated species from the cells through an upregulated cellular autophagy process.…”

Modulation of Amyloid Protein Fibrillation by Synthetic Polymers: Recent Advances in the Context of Neurodegenerative Diseases

“…From the past few years, amino acid-based polymers have attracted remarkable attention of researchers due to their unique physicochemical behavior and widespread applications in biomedical fields . Several amino acid-based polymers have been synthesized from our group, and their promising applications in various field have been explored. , Most recently, we have emphasized the potential role of side chain amino acid-based polymers in the field of protein misfolding and aggregation . We have designed Proline (Pro)-based homopolymers and block copolymers via RAFT polymerization technique (Figure A), and interestingly all the Pro-based polymers are capable of averting the insulin fibrillation process.…”