Structural Optimization of α-Synuclein Fibril Growth Inhibitors

Abstract

Aggregation of alpha-synuclein (αSyn) is involved in the development of Parkinson's disease, a severe neurodegenerative disorder that has no cure at the moment. Recently we showed that bulky molecules binding to the ends of αSyn fibrils can efficiently slowdown fibril growth and developed the first rationally designed inhibitor working on this principle [1]. The constructed fusion protein, consisting of a bulky protein moiety attached to the C-terminus of αSyn molecule, has IC50 of 850 nM. Nevertheless, its inhibitor efficiency can be further improved by increasing steric hindrance and affinity to the fibril end. We studied the structure-activity relationship in a series of inhibitors focusing on the structure, size, and position of the bulky group. We prepared 12 protein-based inhibitors varying: a) sequence of αSyn moiety to improve binding of inhibitor to the fibril end; b) structure of bulky moieties; c) attachment position of the bulky group to αSyn to improve steric hindrance. Their inhibition activities were tested in a kinetic ThT-based assay and compared by IC50 values. Decreasing the electrostatic repulsion between the fibril end and inhibitor molecule by deleting αSyn C-terminus or neutralizing its charge significantly improved inhibitor efficiency (IC50=500 nM and 700 nM, respectively). Changing the bulky group structure and positioning it at the N-terminus allowed us to prepare an inhibitor with IC50 340 nM, that is the most active αSyn fibril growth inhibitor to the best of our knowledge. Our structure-activity relationship studies provide background for rational design of protein-based inhibitors of αSyn fibrillization targeting ends of fibrils and having potential to reach low nanomolar IC50. [1] V V Shvadchak, K Afitska, D A Yushchenko, Angew Chem Int Ed Engl, 2018, 57, 5690.