Human IL-2Rɑ subunit binding modulation of IL-2 through a decline in electrostatic interactions: A computational and experimental approach.

Arezoo Beig Parikhani,Yeganeh Talebkhan,Mahdi Behdani,Mohammad Ali Shokrgozar,Kowsar Bagherzadeh,Sirous Zeinali,Farhad Riazi Rad,Reza Ahangari Cohan,Alireza Biglari,Rada Dehghan,Soheila Ajdary,Michael Massiah

doi:10.1371/journal.pone.0264353

Abstract

Although high-dose IL-2 has clear antitumor effects, severe side effects like severe toxicity and activation of Tregs by binding of IL-2 to high-affinity IL-2R, hypotension, and vascular leak syndrome limit its applications as a therapeutic antitumor agent. Here in this study, a rational computational approach was employed to develop and design novel triple-mutant IL-2 variants with the aim of improving IL-2-based immunotherapy. The affinity of the mutants towards IL-2Rα was further computed with the aid of molecular dynamic simulations and umbrella sampling techniques and the obtained results were compared to those of wild-type IL-2. In vitro experiments by flow cytometry showed that the anti-CD25 mAb was able to bind to PBMC cells even after mutant 2 preincubation, however, the binding strength of the mutant to α-subunit was less than of wtIL-2. Additionally, reduction of IL-2Rα subunit affinity did not significantly disturb IL-2/IL2Rβγc subunits interactions.

Highlights

Human Interleukin-2 (IL-2) is a pleiotropic cytokine that plays pivotal roles in immune responses [1]
Molecular dynamic Simulations (MDs) simulations were carried out using GROMACS v5.1.5 software [17] with GROMOS AMBER force field [18] on the native and mutated IL-2 [mutant 1 (M1) and mutant 2 (M2)] as well as M1 and M2 structures in complex with IL-2Rαβγc, for the best-ranked models obtained from the molecular docking studies
The results obtained from PRODIGY showed that 8 out of 9 single amino-acid substitutions increased binding energy relative to Wild-type human interleukin-2 (wtIL-2), among which K35A, F42A, and E61A resulted in higher binding free energies and eventually larger reduction in binding affinity for IL-2Rα in comparison to other mutations and wtIL-2

Summary

Introduction

Human Interleukin-2 (IL-2) is a pleiotropic cytokine that plays pivotal roles in immune responses [1]. IL-2 is a 15-kDa glycoprotein produced primarily by activated CD4+ and CD8+ T lymphocytes [3] This cytokine exerts its effect by binding to a heterotrimeric receptor on the surface of immune cells. Based on the administered dose of IL-2, it can act as a promoter of both immunosuppression via Tregs, and immune stimulation via other CD4+, CD8+ T, and NK cells [7] This dual effect of IL-2 on the immune response limits its applications as a therapeutic antitumor agent [8]. High affinity IL-2R on Tregs can compete more effectively for IL-2 at low levels, HD-IL-2 can activate even resting cytotoxic lymphocytes and it is used for immune-stimulatory and antitumor activity [9]. In the current study, we designed and tested a novel triple-mutant IL-2 variant with a reduced affinity to the IL-2Rα subunit using a computational approach and experimental analysis

Computational studies

Experimental studies

Discussions

Conclusions