Structure and function of anhydride-modified forms of human insulin: In silico, in vitro and in vivo studies.
Insulin is a therapeutic protein whose amyloid formation is reported in diabetic patients. Four anhydride compounds were used in the current study in order to experiment their potential reducing effect on insulin propensity to form amyloid fibrils. The modified forms (obtained with succinic-, 3,3-dimethylglutaric, 2-phenylglutaric-, and (2-Dodecen-1-yl) succinic anhydride), were first characterized with regard to melting temperature (Tm), changes in secondary structure percentage and hydrophobic surface. Fibril formation was then assessed by Congo red absorbance kinetics and transmission electron microscopy. Functionality was investigated with the use of an insulin tolerance test in NMRI mice. Finally, 10ns molecular dynamics simulations were performed during which structural changes, potential energy, gyration radius, RMSD, and accessible surface area were monitored. In all cases, α-helical structure content of the modified forms was reduced, but thermal stability and structural compactness of modified insulin were increased except in case of the dodecenylated species. All modified insulin forms undergo amorphous aggregation instead of amyloid fibrils formation, and dodecenylated insulin makes the largest amorphous aggregates. In silico results were overall in accordance with in vitro studies. Finally, only succinylated insulin was functional, although dimethylglutaric-modified insulin started to show some activity after 2h.
Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Clinical Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran; Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran.
Maryam
M
Chinisaz
Biosensor Research Center, Endocrinology and Metabolism Molecular-Cellular Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran. Electronic address: aehabibi@sina.tums.ac.ir.
Azadeh
A
Ebrahim-Habibi
Department of Pharmacology, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran; Experimental Medicine Research Center, Tehran University of Medical Sciences, Tehran, Iran.
Ahmad-Reza
AR
Dehpour
Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran.
Parichehreh
P
Yaghmaei
Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran.
Kazem
K
Parivar
Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran.
Ali Akbar
AA
Moosavi-Movahedi
D000812:Anhydrides / Q000737:chemistry* / Q000494:pharmacology*; D000818:Animals; D001786:Blood Glucose / Q000187:drug effects* / Q000378:metabolism; D003198:Computer Simulation; D006801:Humans; D007328:Insulin / Q000737:chemistry* / Q000494:pharmacology*; D008297:Male; D051379:Mice; D011897:Random Allocation; D013329:Structure-Activity Relationship
D000812:Anhydrides; D001786:Blood Glucose; D007328:Insulin
Aggregation; Amyloid fibrils; Anhydride derivatives; Insulin; Mice